Artificial genes for use as controls in gene expression analysis systems

ABSTRACT

Method of producing universal controls for use in gene expression analysis systems such as macroarrays, real-time PCR, northern blots, SAGE and microarrays. The controls are generated either from near-random sequence of DNA, or from intergenic or intronic regions of a genome. Twenty-three specific control sequences are also disclosed. Also presented are methods of using these controls, including as negative controls, positive controls, and as calibrators of a gene expression analysis system.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patent application Ser. No. 10/140,545, filed May 7, 2002, which claims priority to U.S. provisional patent application Nos. 60/289,202, filed May 7, 2001, and 60/312,420, filed Aug. 15, 2001. This application also claims priority to U.S. provisional patent application Ser. Nos. 60/335,115, filed Oct. 24, 2001, and 60/391,367, filed Jun. 25, 2002, the disclosures of which are incorporated herein by reference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ON COMPACT DISC

[0002] The present application includes a Sequence Listing filed on one CD-R disc, provided in duplicate, containing a single file named pto_PB0181.txt, having 56 kilobytes, last modified on Oct. 21, 2002, and recorded on Oct. 21, 2002. The Sequence Listing contained in said file on said disc is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates to a method of using artificial genes as universal controls in gene expression analysis systems. More particularly, the present invention relates to a method of producing universal Controls for use in gene expression analysis systems such as macroarrays, real-time PCR, northern blots, SAGE and microarrays, such as those provided in the Microarray ScoreCard system.

[0005] 2. Description of Related Art

[0006] Gene expression profiling is an important biological approach used to better understand the molecular mechanisms that govern cellular function and growth. Microarray analysis is one of the tools that can be applied to measure the relative expression levels of individual genes under different conditions. Microarray measurements often appear to be systematically biased, however, and the factors that contribute to this bias are many and ill-defined (Bowtell, D. L., Nature Genetics 21, 25-32 (1999); Brown, P. P. and Botstein, D., Nature Genetics 21, 33-37 (1999)). Others have recommended the use of “spikes” of purified mRNA at known concentrations as controls in microarray experiments. Affymetrix includes several for use with their GeneChip products. In the current state of the art, these selected genes are actual genes selected from very distantly related organisms. For example, the human chip (designed for use with human mRNA) includes control genes from bacterial and plant sources.

[0007] Each of the prior art controls consists of transcribed sequences of DNA from some source. As a result, that source cannot be the subject of a hybridization experiment using those controls due to the inherent hybridization of the controls to its source. In addition, the lack of universal references consistent from experiment to experiment and from species to species greatly reduces the ability for scientists to compare data across labs, users, or time. What is needed, therefore, is a set of universal controls that do not hybridize with the DNA of any source which may be the subject of an experiment. More desirably, there is a need for a universal control for gene expression analysis which do not hybridize with any known source.

SUMMARY OF THE INVENTION

[0008] Accordingly, this invention provides a process of producing universal controls that are useful in gene expression analysis systems designed for any species and which can be tested to insure lack of hybridization with mRNA from sources other than the control DNA itself.

[0009] The invention relates in a first embodiment to a process for producing at least one universal control for use in a gene expression analysis system. The process comprises selecting at least one non-transcribed (preferably intergenic, also intronic) region of genomic DNA from a known sequence, designing primer pairs for said at least one non-transcribed region and amplifying said at least one non-transcribed region of genomic DNA to generate corresponding double stranded DNA, then cloning said double stranded DNA using a vector to obtain additional double stranded DNA and formulating at least one control comprising said double stranded DNA.

[0010] The present invention relates in a second embodiment to a process of producing at least one universal control for use in a gene expression analysis system wherein testing of said at least one non-transcribed region to ensure lack of hybridization with mRNA from sources other than said at least one non-transcribed region of genomic DNA is performed.

[0011] The present invention in a third embodiment relates to said process further comprising purifying said DNA and mRNA, determining the concentrations thereof and formulating at least one control comprising said DNA or of said mRNA at selected concentrations and ratios.

[0012] Another embodiment of the present invention is a universal control for use in a gene expression analysis system comprising a known amount of at least one DNA generated from at least one non-transcribed region of genomic DNA from a known sequence, or comprising a known amount of at least one mRNA generated from DNA generated from at least one non-transcribed region of genomic DNA from a known sequence. The present invention may optionally include generating mRNA complementary to said DNA and formulating at least one control comprising said mRNA, by optionally purifying said DNA and mRNA, determining the concentrations thereof and formulating at least one control comprising said DNA or of said mRNA at selected concentrations and ratios.

[0013] Another embodiment of the present invention is a universal control for use in a gene expression analysis system wherein a known amount of at least one DNA sequence generated from at least one non-transcribed region of genomic DNA from a known sequence, a known amount of at least one mRNA generated from DNA generated from at least one non-transcribed region of genomic DNA from a known sequence is included, and the aforementioned control wherein, said DNA and mRNA do not hybridize with any DNA or mRNA from a source other than the at least one non-transcribed region of genomic DNA.

[0014] The present invention, relates to a method of using said universal control, as a negative control in a gene expression analysis system by adding a known amount of said control containing a known amount of DNA, to a gene expression analysis system as a control sample and subjecting the sample to hybridization conditions in the absence of complementary labeled mRNA and examining the control sample for the absence or presence of signal.

[0015] Further, said controls can be used in a gene expression analysis system by adding a known amount of a said control containing a known amount of DNA to a gene expression analysis system as a control sample and subjecting the sample to hybridization conditions, in the presence of a said control containing a known amount of labeled complementary mRNA, and measuring the signal values for the labeled mRNA and determining the expression level of the gene transcript based on the signal value of the labeled mRNA.

[0016] Additionally, said controls may be used as calibrators in a gene expression analysis system by adding a known amount of a said control containing known amounts of several DNA sequences to a gene expression analysis system as control samples and subjecting the samples to hybridization conditions in the presence of a said control containing known amounts of corresponding complementary labeled mRNAs, each mRNA being at a different concentration and measuring the signal values for the labeled mRNAs and constructing a dose-response or calibration curve based on the relationship between signal value and concentration of each mRNA.

[0017] Also, the present invention relates to a method of using said controls as calibrators for gene expression ratios in a two-color gene expression analysis system by adding a known amount of at least one of said controls containing a known amount of DNA to a two-color gene expression analysis system as control samples and subjecting the samples to hybridization conditions in the presence of a said control containing known amounts of two differently labeled corresponding complementary labeled mRNAs for each DNA sample present and measuring the ratio of the signal values for the two differently labeled mRNAs and comparing the signal ratio to the ratio of concentrations of the two or more differently labeled mRNAs.

[0018] A further embodiment of the present invention is a process of producing controls that are useful in gene expression analysis systems designed for any species and which can be tested to insure lack of hybridization with mRNA from sources other than the synthetic sequences of DNA from which the control is produced.

[0019] One or more such controls can be produced by a process comprising synthesizing a near-random sequence of non-transcribed DNA, designing primer pairs for said at least one near random sequence and amplifying said non-transcribed DNA to generate corresponding double stranded DNA, then cloning said double stranded DNA using a vector to obtain additional double stranded DNA and formulating at least one control comprising said double stranded DNA.

[0020] The process can also be used to produce at least one control for use in a gene expression analysis system wherein testing of said sequence of non-transcribed synthetic DNA to ensure lack of hybridization with mRNA from sources other than said sequence of non-transcribed DNA is performed.

[0021] Additionally, mRNA complementary to said synthetic DNA can be generated and formulated to generate at least one control comprising said mRNA.

[0022] DNA and mRNA can be subsequently purified, the concentrations thereof determined, and one or more controls comprising said DNA or said mRNA at selected concentrations and ratios be formulated.

[0023] Another embodiment of the present invention is a control for use in a gene expression analysis system produced by the process comprises synthesizing a near-random sequence of DNA, designing primer pairs for said synthetic DNA and amplifying said DNA to generate corresponding double stranded DNA, then cloning said double stranded DNA using a vector to obtain additional double stranded DNA and formulating at least one control comprising a known amount of at least one said double stranded DNA or a known amount of at least one mRNA generated from said DNA, and optionally, wherein, said DNA and mRNA do not hybridize with any DNA or mRNA from a source other than said DNA sequence of non-transcribed DNA.

[0024] The present invention, additionally, relates to a method of using said controls containing a known amount of DNA, as a negative control in a gene expression analysis system including adding a known amount of said control containing a known amount of DNA to a gene expression analysis system as a control sample, and subjecting the sample to hybridization conditions in the absence of complementary labeled mRNA and examining the control sample for the absence or presence of signal.

[0025] Further, said controls may be used in a gene expression analysis system wherein a known amount of a said control containing a known amount of DNA is added to a gene expression analysis system as a control sample and subjecting the sample to hybridization conditions in the presence of a said control containing a known amount of labeled complementary mRNA and measuring the signal values for the labeled mRNA and determining the expression level of the gene transcript based on the signal value of the labeled mRNA.

[0026] The present invention, also relates to a method of using said controls as calibrators in a gene expression analysis system including adding known amounts of a said control containing known amounts of several DNAs to a gene expression analysis system as control samples and subjecting the samples to hybridization conditions in the presence of a said control containing known amounts of corresponding complementary labeled mRNAs, each mRNA being at a different concentration and measuring the signal values for the labeled mRNAs and constructing a dose-response or calibration curve based on the relationship between signal value and concentration of each mRNA.

[0027] The present invention, additionally, relates to a method of using said controls as calibrators for gene expression ratios in a two-color gene expression analysis system comprising adding a known amount of at least one of said controls containing a known amount of DNA to a two-color gene expression analysis system as control samples and subjecting the samples to hybridization conditions in the presence of a said control containing known amounts of two differently labeled corresponding complementary labeled mRNAs for each DNA sample present and measuring the ratio of the signal values for the two differently labeled mRNAs and comparing the signal ratio to the ratio of concentrations of the two or more differently labeled mRNAs.

[0028] Further embodiments and uses of the current invention will become apparent from a consideration of the ensuing description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The above and other objects and advantages of the present invention will be apparent upon consideration of the following detailed description taken in conjunction with the accompanying drawings, in which like characters refer to like parts throughout, and in which:

[0030]FIG. 1 shows representative results for the selection of universal controls that do not cross-hybridize with human RNA;

[0031]FIG. 2 shows representative results for the selection of universal controls that do not cross-hybridization with each other;

[0032]FIG. 3 represents a performance evaluation of the universal controls;

[0033]FIG. 4 shows a scatter plot of raw signals for the calibration and ratio controls from a two-color hybridization experiment;

[0034]FIG. 5 shows calibration curves based on the Calibration controls for a representative hybridization experiment;

[0035]FIG. 6 presents the control nucleotide sequence of DR1 (SEQ ID NO: 1);

[0036]FIG. 7 presents the control nucleotide sequence of DR2 (SEQ ID NO: 2);

[0037]FIG. 8 presents the control nucleotide sequence of DR3 (SEQ ID NO: 3);

[0038]FIG. 9 presents the control nucleotide sequence of DR4 (SEQ ID NO: 4);

[0039]FIG. 10 presents the control nucleotide sequence of DR5 (SEQ ID NO: 5);

[0040]FIG. 11 presents the control nucleotide sequence of DR6 (SEQ ID NO: 6);

[0041]FIG. 12 presents the control nucleotide sequence of DR7 (SEQ ID NO: 7);

[0042]FIG. 13 presents the control nucleotide sequence of DR8 (SEQ ID NO: 8);

[0043]FIG. 14 presents the control nucleotide sequence of DR9 (SEQ ID NO: 9);

[0044]FIG. 15 presents the control nucleotide sequence of DR10 (SEQ ID NO: 10);

[0045]FIG. 16 presents the control nucleotide sequence of RC1 (SEQ ID NO: 11);

[0046]FIG. 17 presents the control nucleotide sequence of RC2 (SEQ ID NO: 12);

[0047]FIG. 18 presents the control nucleotide sequence of RC3 (SEQ ID NO: 13);

[0048]FIG. 19 presents the control nucleotide sequence of RC4 (SEQ ID NO: 14);

[0049]FIG. 20 presents the control nucleotide sequence of RC5 (SEQ ID NO: 15);

[0050]FIG. 21 presents the control nucleotide sequence of RC6 (SEQ ID NO: 16);

[0051]FIG. 22 presents the control nucleotide sequence of RC7 (SEQ ID NO: 17);

[0052]FIG. 23 presents the control nucleotide sequence of RC8 (SEQ ID NO: 18);

[0053]FIG. 24 presents the control nucleotide sequence of Utility1 (SEQ ID NO: 19);

[0054]FIG. 25 presents the control nucleotide sequence of Utility2 (SEQ ID NO: 20);

[0055]FIG. 26 presents the control nucleotide sequence of Utility3 (SEQ ID NO: 21);

[0056]FIG. 27 presents the control nucleotide sequence of Negative1 (SEQ ID NO: 22);

[0057]FIG. 28 presents the control nucleotide sequence of Negative2 (SEQ ID NO: 23);

[0058]FIG. 29 presents the nucleotide sequence of DR1s used in a spike mix (SEQ ID NO: 24);

[0059]FIG. 30 presents the nucleotide sequence of DR2s used in a spike mix (SEQ ID NO: 25);

[0060]FIG. 31 presents the nucleotide sequence of DR3s used in a spike mix (SEQ ID NO: 26);

[0061]FIG. 32 presents the nucleotide sequence of DR4s used in a spike mix (SEQ ID NO: 27);

[0062]FIG. 33 presents the nucleotide sequence of DR5s used in a spike mix (SEQ ID NO: 28);

[0063]FIG. 34 presents the nucleotide sequence of DR6s used in a spike mix (SEQ ID NO: 29);

[0064]FIG. 35 presents the nucleotide sequence of DR7s used in a spike mix (SEQ ID NO: 30);

[0065]FIG. 36 presents the nucleotide sequence of DR8s used in a spike mix (SEQ ID NO: 31);

[0066]FIG. 37 presents the nucleotide sequence of DR9s used in a spike mix (SEQ ID NO: 32);

[0067]FIG. 38 presents the nucleotide sequence of DR10s used in a spike mix (SEQ ID NO: 33);

[0068]FIG. 39 presents the nucleotide sequence of RC1s used in a spike mix (SEQ ID NO: 34);

[0069]FIG. 40 presents the nucleotide sequence of RC2s used in a spike mix (SEQ ID NO: 35);

[0070]FIG. 41 presents the nucleotide sequence of RC3s used in a spike mix (SEQ ID NO: 36);

[0071]FIG. 42 presents the nucleotide sequence of RC4s used in a spike mix (SEQ ID NO: 37);

[0072]FIG. 43 presents the nucleotide sequence of RC5s used in a spike mix (SEQ ID NO: 38);

[0073]FIG. 44 presents the nucleotide sequence of RC6s used in a spike mix (SEQ ID NO: 39);

[0074]FIG. 45 presents the nucleotide sequence of RC7s used in a spike mix (SEQ ID NO: 40);

[0075]FIG. 46 presents the nucleotide sequence of RC8s used in a spike mix (SEQ ID NO: 41);

[0076]FIG. 47 presents the nucleotide sequence of Utility1s used in a spike mix (SEQ ID NO: 42);

[0077]FIG. 48 presents the nucleotide sequence of Utility2s used in a spike mix (SEQ ID NO: 43);

[0078]FIG. 49 presents the nucleotide sequence of Utility3s used in a spike mix (SEQ ID NO: 44);

[0079]FIG. 50 presents the nucleotide sequence of Negative1s used in a spike mix (SEQ ID NO: 45); and

[0080]FIG. 51 presents the nucleotide sequence of Negative2s used in a spike mix (SEQ ID NO: 46).

DETAILED DESCRIPTION OF THE INVENTION

[0081] The present invention teaches universal Controls for use in gene expression analysis systems such as microarrays. Many have expressed interest in being able to obtain suitable genes and spikes as controls for inclusion in their arrays.

[0082] An advantage of the universal Controls of this invention is that a single set can be used with assay systems designed for any species, as these Controls will not be present unless intentionally added. This contrasts with the concept of using genes from “distantly related species.” For example, an analysis system directed at detecting human gene expression might employ a Bacillus subtilis gene as control, which may not be present in a human genetic material. But this control might be present in bacterial genetic material (or at least, cross hybridize), thus it may not be a good control for an experiment on bacterial gene expression. The novel universal Controls presented here provide an advantage over the state of the art in that the same set of controls can be used without regard to the species for the test sample RNA.

[0083] The present invention employs the novel approaches of using either non-transcribed genomic sequences or totally random synthetic sequences as a template and generating both DNA and complementary “mRNA” from such sequences, for use as controls. The Controls could be devised de novo by designing near-random sequences and synthesizing them resulting in synthetic macromolecules as universal controls. Totally synthetic random DNA fragments are so designed that they do not cross-hybridize with each other or with RNA from any biologically relevant species (meaning species whose DNA or RNA might be present in the gene expression analysis system). The cost of generating such large synthetic DNA molecules can be high. However, they only need to be generated a single time. Additionally, fragment size can be increased by ligating smaller synthetic fragments together by known methods. In this way, fragments large enough to be easily cloned can be created. Through cloning and PCR sufficient quantities of DNA for use as controls can be produced and mRNA can be generated by in vitro transcription for use in controls.

[0084] A simpler approach is to identify sequences from the intergenic or intronic regions (referred here as non-transcribed regions) of genomic DNA from an organism, and use these as a template for synthesis via PCR (polymerase chain reaction). Ideally, sequences of around 1000 bases (could range from 500 to 2000 bases) are selected based on computer searches of publicly accessible sequence data. The criteria for selection include:

[0085] 1. The sequence must be from a non-transcribed region; and

[0086] 2. The sequence must not have homology with or be predicted to hybridize with any known/published gene or expressed sequence tag (EST).

[0087] PCR primer pairs are designed for the selected sequence(s) and PCR is performed using genomic DNA (as a template) to generate PCR fragments (double strand DNA) corresponding to the non-transcribed sequence(s) as the control DNA. Additional control DNA can be cloned using a vector and standard techniques. Subsequently, standard techniques such as in vitro transcription are used to generate mRNA (complementary to the cDNA and containing a poly-A tail) as the control mRNA. Standard techniques are used for purifying the Control DNA and Control mRNA products, and for estimating their concentrations.

[0088] Empirical testing is also performed to ensure lack of hybridization between the Control DNA on the array and other mRNAs, as well as with mRNA from important gene expression systems (e.g., human, mouse, Arabidopsis, etc.).

[0089] The above approaches were used to generate twenty-three universal control sequences from intergenic regions of the yeast Saccharomyces cerevisiae genome. Specifically, using yeast genome sequence data publicly available (http://genome-www.stanford.edu/Saccharomyces/), intergenic regions approximately 1 kb in size were identified. These sequences were BLAST'd and those showing no homology to other sequences were identified as candidates for artificial gene controls. Candidates were analyzed for GC-content and a subset with a GC-content of ≧36% was identified. Specific primer sequences have been identified and primers synthesized. PCR products amplified with the specific primers have been cloned directly into the PGEM™-T Easy vector (Promega Corp., Madison, Wis.). Both array targets and templates for spike mRNA have been amplified from these clones using distinct and specific primers.

[0090] A greater number of intergenic regions have been cloned for testing. DNA samples from all the candidates were amplified, spotted on glass microarray slides and hybridized with mRNA samples from several species and each candidate spike mRNA, respectively, to identify those that do not cross-hybridize. First, they were screened for no cross-hybridization with RNA from different biological species. mRNA from human (eight tissues: skeletal muscle, spleen, liver, heart, kidney, brain, placenta and lung), mouse (six tissues: skeletal muscle, spleen, liver, heart, kidney and brain), rat (six tissues: skeletal muscle, spleen, liver, heart, kidney and brain), yeast (S. cerevisiae) and bacteria (E. coli and two Archaea species), as well as total RNA from plant (Arabidopsis, Oil Palm) were tested against the control candidates. Candidates that did not cross-react with the RNA samples from the species tested were then selected for cross-hybridization with each other. The candidates were hybridized with each candidate mRNA independently.

[0091] From the candidate clones that exhibited specific hybridization, twenty-three were included into the final set of universal controls. FIG. 6 through FIG. 28 presents the nucleotide sequences of the twenty-three controls spotted on the microarray slides, while FIGS. 29 through 51 presents the nucleotide sequences of the twenty-three controls that were transcribed and used in a spike mix, respectively. SEQ ID NO: 1 through SEQ ID NO: 23 present the nucleotide sequences of the twenty-three controls spotted on the microarray slides, while SEQ ID NO: 24 through SEQ ID NO: 46 present the nucleotide sequences of the controls that were transcribed and used in a spike mix.

[0092] These universal controls, when included in microarray experiments, perform as:

[0093] 1. Negative controls: Control DNA included in the array, but for which no complementary artificial mRNA is spiked into the RNA sample, serves as a negative control;

[0094] 2. Calibration controls: Several different Control DNA samples may be included in an array, and the complementary Control mRNA for each is included at a known concentration, each having a different concentration of mRNA. The signals from the array features corresponding to these Controls or Calibrators may be used to construct a “dose-response curve” or calibration curve to estimate the relationship between signal and amount of mRNA from the sample;

[0095] 3. Ratio controls: In two-color microarray gene expression studies, it is possible to include different, known, levels of Control mRNA complementary to Control DNA in the labeling reaction for each channel. The ratio of signals for the two dyes from a particular gene can be compared to the ratio of signals from the two dyes of the Control mRNA. This can serve as a test of the accuracy of the system for determining gene expression ratios.

[0096] 4.Utility controls: These controls can be added into the sample preparation steps (such as RNA extraction and purification) for normalization of the biological samples and assessment of sample losses during preparation. Alternatively, they can be added to labeling reactions as additional calibrators or ratios.

[0097] Mixtures of several different Control mRNA species can be prepared (spike mixes) at known concentrations and ratios to simplify and standardize the experimental protocol while providing a comprehensive set of precision and accuracy information. Table 1 demonstrates one embodiment of this concept. The mRNA from the final set of clones have been pre-mixed at specific concentrations and ratios so they can serve as the various controls when hybridized to their corresponding control DNA spotted on the arrays. Ten calibrators (those included in the labeling reaction at a ratio of 1:1) spanning a dynamic range of 4.5 orders of magnitude are included as calibration controls. Eight ratio controls are included, at two expression levels (low and medium to high) and reversed with respect to the reference and test samples.

[0098] The universal controls as shown in Table 1 can be used as references for microarray validation and standardization across biological species and experimental platforms. These controls can be used to verify the accuracy and precision of gene expression ratios, and the sensitivity and dynamic range of the microarray system. Through the use of Calibration (standard) curves, these controls may allow reporting gene expression levels in consistent mass units, improving the comparisons of results across laboratories.

[0099] The following examples demonstrate how these Control DNA and Control mRNA were generated, and then used as universal controls in microarray gene expression experiments. They are representative of the many different types of experiments that could benefit from the use of these controls. The following examples are offered by way of illustration and not by way of limitation. TABLE 1 Suggested Control mRNA spike mix composition for two-color gene expression ratio experiments. Target mRNA in the Spike Mix Control Control Cy3:Cy5 (pg/2 μl of spike) Type Name Ratio Cy3 Cy5 Calibration DR1s 1:1 30 000 30 000 Calibration DR2s 1:1 10 000 10 000 Calibration DR3s 1:1 3 000  3 000 Calibration DR4s 1:1 1 000  1 000 Calibration DR5s 1:1   300   300 Calibration DR6s 1:1   100   100 Calibration DR7s 1:1    30    30 Calibration DR8s 1:1    10    10 Calibration DR9s 1:1    3    3 Calibration DR10s 1:1    1    1 Ratio RC1s  3:1 low   300   100 Ratio RC2s  1:3 low   100   300 Ratio RC3s  3:1 high  3 000  1 000 Ratio RC4s  1:3 high  1 000  3 000 Ratio RC5s 10:1 low   300    30 Ratio RC6s  1:10 low    30   300 Ratio RC7s 10:1 high 10 000  1 000 Ratio RC8s  1:10 high  1 000 10 000 Utility Utility1s User User User defined defined defined Utility Utility2s User User User defined defined defined Utility Utility3s User User User defined defined defined Negative Negative1s NA    0    0 Negative Negative2s NA    0    0

EXAMPLE 1 Generation of Artificial Controls from Intergenic Regions of S. cerevisiae Genome

[0100] Using yeast genomic sequence data publicly available (http://genome-www.stanford.edu/Saccharomyces/), intergenic regions (YIRs) approximately 1 kb in size were identified. These sequences were BLAST'd and those showing no homology to other sequences were identified as candidates for artificial gene controls. Candidates were analyzed for GC-content and a subset with a GC-content of ≧36% was identified. Specific primer sequences have been identified and synthesized. PCR products amplified with the specific primers have been cloned directly into the pGEM™-T Easy vector (Promega Corp., Madison, Wis.). Both array targets and templates for spike mRNA have been amplified from these clones using distinct and specific primers.

[0101] When used as DNA controls, the YIR sequences were amplified by PCR with specific primers, using 5 ng of cloned template (plasmid DNA) and a primer concentration of 0.5 μM in a 100 μl reaction volume, and cycled as follows: 35 cycles of 94° C. 20 sec., 52° C. 20 sec., 72° C. 2 min., followed by extension at 72° C. for 5 min.

[0102] All YIR control mRNAs for the spike mix are generated by in vitro transcription. Templates for in vitro transcription (IVT) are generated by amplification with specific primers that are designed to introduce a T7 RNA polymerase promoter on the 5′ end and a polyT (T21) tail on the 3′ end of the PCR products. Run-off mRNA is produced using 1 μl of these PCR products per reaction with the AmpliScribe system (Epicentre, Madison, Wis.). IVT products are purified using the RNAEasy system (Qiagen Inc., Valencia, Calif.) and quantified by spectrophotometry.

[0103] Initially, fifty intergenic region sequences have been cloned for testing. DNA samples from all the candidates were amplified, spotted on glass microarray slides and hybridized with mRNA samples from several species and each candidate spike mRNA, respectively, to identify those that do not cross-hybridize. First, they were screened for no cross-hybridization with RNA from different biological species. mRNA from human (8 tissues: skeletal muscle, spleen, liver, heart, kidney, brain, placenta and lung), mouse (6 tissues: skeletal muscle, spleen, liver, heart, kidney and brain), rat (6 tissues: skeletal muscle, spleen, liver, heart, kidney and brain), yeast (S. cerevisiae) and bacteria (E. coli and two Archaea species), as well as total RNA from plant (Arabidopsis, Oil Palm) were tested against the control candidates.

[0104]FIG. 1 shows the hybridization of candidates with human brain mRNA. The results indicated that two YIR clones, 33 and 62, hybridized with human brain RNA while the other candidates did not (since no appreciable signal is detected). Clones, such as 33 and 62, that exhibited such cross-hybridization were removed from the set of candidates for universal controls.

[0105] Candidates that did not cross-react with the RNA samples from the species tested were then tested for cross-hybridization with each other. The candidates were hybridized with each candidate mRNA independently. In FIG. 2 the labeled mRNA made from clone #50 was specifically hybridized against all other candidate clones. It hybridized only to its corresponding target DNA and can be included into the candidate set. However, clone #52 bound to the spot of clone #49 besides its own and therefore was not included in the candidate set.

[0106] From the candidate clones that exhibited specific hybridization, twenty-three are included into the final set of universal controls. FIG. 6 through FIG. 28 presents the nucleotide sequences of the twenty-three controls spotted on the microarray slides, while FIGS. 29 through 51 presents the nucleotide sequences of the twenty-three controls as used in a spike mix, respectively. The sequences of these clones are further presented in the Sequence Listing, incorporated herein by reference in its entirety, as follows:

[0107] SEQ ID NOs: 1-23 (nt, control nucleotide sequences, including calibration controls 1 through 10, ratio controls 1 through 8, utility controls 1 through 3, and negative controls 1 and 2 respectively);

[0108] SEQ ID NOs: 24-46 (nt, spike mix nucleotide sequences, including calibration controls 1 through 10, ratio controls 1 through 8, utility controls 1 through 3, and negative controls 1 and 2 respectively);

[0109] Upon confirmation of the exact structure, each of the above-described nucleic acids of confirmed structure is recognized to be immediately useful as a control.

EXAMPLE 2 Performance Evaluation of the Artificial Controls

[0110] The universal controls (both the spike mixes and their corresponding spotting samples) have been evaluated for their performance in real microarray experiments and tested for the following.

[0111] Experimental design, including array design and the hybridization sample concentration were tested (FIG. 3). Control samples were spotted in five replicates and hybridized with probes prepared with the spike mix only or the spike mixes with skeletal muscle mRNA. The same array image in FIG. 3 is shown at two different gray scales for easy visualization of signals across the entire dynamic range.

[0112] Universal utility, including hybridization of the spikes on pre-arrayed slides from various species were also tested. The controls showed no cross-hybridization on human, rat, mouse, Arabidopsis, Yeast and E. coli pre-arrayed slides from commercial sources (data not shown).

[0113] Spike mix performance was tested, including ratio performance and Calibration curves (FIGS. 4 and 5). The mRNA from the final set of clones have been pre-mixed at specific concentrations and ratios (see Table 1 above) so they can serve as the various controls when hybridized to their corresponding control DNA spotted on the arrays. Ten calibrators (those included in the labeling reaction at a ratio of 1:1), spanning a dynamic range of 4.5 orders of magnitude, are included as calibration controls. Eight ratio controls are included, at two expression levels (low and medium to high) and reversed with respect to the reference and test samples.

[0114]FIG. 4 shows a scatter plot of raw signals for the calibration and ratio controls from a two-color hybridization experiment. The Calibrators are accurately and precisely clustered at the 45-degree line and the ratios at their expected target values at high (labeled ‘H’) and low (labeled ‘L’) levels of expression.

[0115]FIG. 5 shows calibration curves based on the Calibration controls for a hybridization experiment. In this “standard curve”, the Cy3 and Cy5 signals from the calibration controls are plotted as a function of the amount of mRNA in the spike mix. The error bars represent the 95% confidence intervals for the mean value. From such curves, attributes such as the limit of detection, the linear dynamic range and the signal saturation limit can be assessed. The application of the universal controls for the generation of standard curves can be the first step towards true quantitation of expression levels from microarray experiments.

[0116] The controls as shown in Table 1 can be used as references for microarray validation and standardization across biological species and experimental platforms. These controls can be used to verify the accuracy and precision of gene expression ratios, and the sensitivity and dynamic range of the microarray system. Through the use of Calibration (standard) curves, these controls may allow reporting gene expression levels in consistent mass units, improving the comparisons of results across laboratories

[0117] The above examples illustrate specific aspects of the present invention and are not intended to limit the scope thereof in any respect and should not be so construed.

[0118] Those skilled in the art having the benefit of the teachings of the present invention as set forth above, can effect numerous modifications thereto. These modifications are to be construed as being encompassed within the scope of the present invention as set forth in the appended claims.

1 46 1 936 DNA Saccharomyces cerevisiae 1 tgttgtccaa gaaaggaggg attttgttca tcagaaaaga attcagaaaa gcaaggaaac 60 agtactatcg tttagaatgt agaatgatag gttgcttgct aattctatta tggcacgaat 120 gatacaccca tattttcaac aaaatcaata cccactagca tcattgagcc aactatttgt 180 caatgcaacc attaccggta cttcatcctg atttaacgag tctacttttt tatcacgtca 240 aaatttactt gttttcctgt aaacccgaaa taaaggcaaa aaagacctgg gtgcaattac 300 gaataaatgt acaataatca tcctgtttgc atagtaaact tccagttaga gtcacacaac 360 gcaatgaatt ttgacagttt tctgtgcgat attctttggt aaacgtaaag aacaggcaac 420 ttttggtaca atggattcta gcccatatgg ttcatttctg gtgcattcgc aaagtcagta 480 tttgtctagc tgtgttttct ggctgagaga cattatgatg ttattcattg ttatggatat 540 ctctgtagct catgctgctt atttctccct aaaaaagttt tttctctcga atacattctt 600 gaccatttca tagtgaaatt cttgtactta tttaaaacca aaaatggaag tattcataca 660 tccccctatc aaaaacactc aataagtttc gaattattcg ttcgtctaaa cagtgtccaa 720 tactcaaagg ggtattcaag acggcacaaa atcagcatct tcccttatcc gtgttccaga 780 aataccacgc taaggttttt cctcctacaa tccataaaat cattaaggag gcagcttgaa 840 aaatcttgaa attcaaaaga gttatcttgg gctaatcgaa attaacgata accagagtag 900 aatattcaag atcacagctc caccttagtt tcgagg 936 2 1000 DNA Saccharomyces cerevisiae 2 gcgaataacc aaaacgagac tactttttac cattacaacc attttctttt tccctatttc 60 tcactggttg acagaaatca gtgtgctatc atcctaccat atgcgctaaa cttattgtct 120 ttctcctcct agagatgctg tattccatgc atattctgaa cgatgggttg gtgtttttat 180 caagcaaggt taatcacatg gcgtggcttg ctccacacat cagtagaaaa cgcataccgc 240 agcggaatcc ttaaataata agtgatttta ctgttcatca actacaatcg gactctttca 300 caattaccct tcttgttttc cacatttact gttaaatgaa gggatgtaca gaaggcttag 360 gaaaacctgt gctgaatact ggatggacac tgcattccca cagtgaaact tttatagata 420 cactgtcagt tattttcgaa ctttcatcaa gttgctgagt tttagtatcc ctttgcctta 480 gctatatgtt tgaatgagca aaatatttgc aatgtctcta gctttcttga aatattggtt 540 tatattgagg gcttggtaag atttcaaatt tcactttgaa atactcagga gaaaaatcat 600 gctcttttga taatttggtg actaaacata cataaaacag tttaattttg ggtggtaatg 660 gctgtgtgac tagctataga aagaaaaaaa ttaaaaaaaa aaaaaaaaat caagtagttc 720 ctgcactgcg acgtccatta tagcattatg aattggtccc tgatttacgc atgcgataaa 780 ctatttttag cgcagccgca tattatccga gaataacttc cgacataaga aaattcgcag 840 aaaatagata aaaaactgct cttggcattc ttcacttcct ctattacaca ctgtgtcata 900 ccacaatcat ctcacagtat gtatttgtat gtttatacat gctataacgt aaaacaatgt 960 agaatatata tctaaatacc tcacggtttt agtttagtgc 1000 3 980 DNA Saccharomyces cerevisiae 3 tgtgagaaat tcactagctt cacctaaaag caaaaaatct caaaattccc aatattcaca 60 tagtctaaag taccgatagc aaccaacata tataaacagt agtattttac gaagctgaat 120 tgcaagatta gtgagaggag aataaccgga taattttttt ggattacgtt attgttaaag 180 gctataatat taggtgaaac agaatgtcct agaagttttt ttctttcatg ttaaatttat 240 tgattcttgc gcttcagctt ttataaaaca taagaactgt ttcttcacgt taacttcttg 300 tgccacatat aatgatgtac tagtaatatg ggtactattt ggcagatgat atttgatttt 360 tattcaagac ggttactgtt tctacgattg atattttcat tcctggatat catcttgcca 420 gatcacttac aatttaggcc gcgcctgaat tgaagagtac ttcaatacgt agtgtactgt 480 ccaaactctc ttccaaattt ttaatattta gctggggttg ggtaacaagt gagcaaggga 540 aaaagtgaac attttaagaa gaacaataaa atagcaagag atggaatggt aatgcttggc 600 tctcgagaag agtagcataa aacgagactt gtttaaaaca ggatatgaca tacttcaatt 660 cagctttccc tatcagccgc tcgagcagtt atataggtgt gttgccggag taatttggcg 720 gaggccaaca gtggctaggc ggcaacgcct ggaacacgcg cttaaaagtt ctggaaggtt 780 cgcgaattga gaactgctca ggggcgaata caggggcggc cttggcggca ggggggaggc 840 ctctgtgaag ttagttatat aagacttgct gtcatcgttt ttttgatccc ggcaggaact 900 atcttttatt ctcatacata cggtcaagaa gtataattat acataacata gggacacgtt 960 caggcaattg tccatatccc 980 4 937 DNA Saccharomyces cerevisiae 4 gatgtctgtt ttctttatgc aggatattaa atacaagttt gtgcttaaga aatttccatg 60 aagatatcaa catttattgt aggaattgca taaatagatg aattatgtgc cgctggacgt 120 ttatagatag cataagcaca atgacttaaa ggttataata ctcattgata tcactctgat 180 tataaaatcg taatatgcga ataggtgaac taatcggaat aacccatacg acacttcaag 240 cttcaattct atttcaactg tagtgcctgc tagtgaagaa tacaaaagta gcatacgtga 300 tgtgcaaaaa atgcgctact tatcacacaa gtaccttgcg caagaagggt actctaaacc 360 ggggccatcg cattaccaga cggagatgta ttctttatga agcaataatt ggaggtgtat 420 caagttcgaa actgctgatg ctatggattt acatctttct tatgcacaag gcttgcttgt 480 gtttctgagt agttagtttt tagatttttg tcaagtctgg ggtaagttaa ttcgagcaaa 540 attaacggca cgttattcta atgcatatgt tgttcatata ttcttttaca aagaggtttg 600 gaatgatgtc accgatgtta gaatgttagg agaatttcat gtgaatttta gtccaagtgt 660 tgaagttctc ttctgcagtt agggcacgta catggcaacg atatcgtttt tgatgtatta 720 atcttagtag gcgttgagtt tgtatgttac ttttctcagg tgatgaagcg tgatgacgat 780 gacaaaaatg ggttataata gggcgcacta tcatcatgcg tgattgatat ttaaccaatg 840 tcttgagtac atcaactcca gaaaatgggt cattatatgc ctagcatgta ttatttgaga 900 cataaagttt tatctcgaga ccttgacgta taggaaa 937 5 940 DNA Saccharomyces cerevisiae 5 ctgtagattt gcatggacgc acgtcgccca tacgccaaac tttggcaatg atactcgtta 60 ttcgtaatat cagtccgtca aggtgctgtg atttctctat tttatattgc ctattatttt 120 ttcaaatgat ttgagccgtt ttaaattgag tatgcaatga gtcttttgaa tcaaccgtaa 180 ggcagttcca taaccactgc cacgaatacg tttcactacc ttgaagaatc tctaatgtag 240 gccgtattct tcgcacttag ttctgacgat gtagacatct cattatataa gagcataagc 300 gcctgtttct agaatcattt cttcgtgacc cagctttttg agttatttcg cggtattttg 360 aaacatttct cgagcttgac gtgaacatcc ttatatttca tgacaaactc gatcattgga 420 acatccctgc ctcgatttta gagctagtat caaatttcaa tctctttgtg atggagcccc 480 gctcctattt caaaagagaa gtttcttgta tgcatatgtt attgaagtct gattatagca 540 agtgcaatgt cgtctcaatt attttaacta tttttagcca tacatgttag ttatcctcaa 600 agagagcctc cagactggga agcagtgttt gtcatttcaa ataagtagat ttcacagttt 660 gtatgatttt cgaagccagg attcattggg ctttgagtaa agagaagccg cgtattacga 720 acagcttacg atattgtaaa atattccctt attgtggtgc cccaatggat acatgccaga 780 gaaatgtctg tgaaattgaa caattacaat gacgagagca agtaatccgg cggccttgtc 840 tctctttcac tagtaccgtc tatatctctt gagcgccaat atgcgaaagc tttcacaagg 900 ttgatgttca tggtattcgg cgtcgatagc gaattgctta 940 6 950 DNA Saccharomyces cerevisiae 6 ttagtttgga acagcagtgt agataccgtc cttggataga gcgctggaga tagctggtct 60 caatctggtg gagtaccatg ggacaccagt gatgactcta gtgacttgat cagcgggaat 120 accagtcaac atagtggtga aatcaccgta gttgaaaaca gcttcagcaa tttcaactgg 180 gtaagtttca gttggatgag cagcttggaa catatagtat tcagccaaat gagctctgat 240 atctgagacg tagacaccta attcgaccag gttaactctt tcgtcagagg gagataaagt 300 agtggtggct ggggcagcag cgacaccagc agcaatagca gcgacaccag caacaattga 360 agttagtttg accatttttt tcgattgaac ttttgtagat ctttttagtg aagatgtgag 420 ctcactcgaa tgtaaataac aatgccaaat tgtcggaaag agttaatcaa agctgctcta 480 tttatatgcc gttttttaat aagcgacgga cgaacagata aattgttgaa tagctatttc 540 actgctgata tttctcttac ttgggctccc ctatcccata ctcttcacca ctacaaatat 600 gcagttgccc tttcttcaac aatgcttttt ttatagatct cgtatacgga tccgcgcctt 660 tgtactacct atatcttatt atgatatata caggagcaca ggaatgttcg gtacagggat 720 gataccttta aaggaagttt tggcatgcct tgacaacttc aattaatctt tggccaagaa 780 aatgaaccag aaatcaaatt ttattctgtg ccctctgaac gagggcaata tccaatgttt 840 gacactaaac ggttgtcagg agaaaaattg aatgtttccc aaatcagaaa cattaaaatc 900 cctctatatg atcagaggag tcgtacctgt tagggtatga gcgaggaaac 950 7 982 DNA Saccharomyces cerevisiae 7 aatgagttac cgtctgttac ttttgggacg gtttttgcac taagaacaga cgagtttacg 60 gttatcctca acaagcaagc aagtatttgc taatctagat gccattccga atcattactc 120 atacgttact attgagagat gttttacaat agatgagaag aatacaatgt ccagagctcc 180 tggtatgcta gagtgcatat tccaggtctt attcgaatca tatcataccg tccatttcaa 240 caatggtgaa atgtggtcca catatatcag aaatcttaac atttagtgag gagagccagt 300 agaaaaatgt gcgcaagcgg aaagaagtca ttcacagaca cgtttaacaa aacaccacca 360 cagcagcttt gtctcttgat tctgatcagt ttgccatcga agaagcaaaa ttgtggtgtt 420 atttttttca aacaaaactt ttttggcaac agcagttttc ttctggatat ttgtacttta 480 tcatccaacc gatgaaagct ggtttcctgt caacctacat ttaaatggcc cgtacttctt 540 caaaaccgct agataagcaa attaacccaa cttttgagcg tcctaaattc cccttggctc 600 agaagactcg ttaatatggg aagtttaagt cctaccatat aatcaaattg gaagctttct 660 gtgttcgaat ggctattcta accgctgggc tattaatcag aggggaagtg aaatgaccga 720 gacgtattat acgtcatgtt gacatcaaca atttaaggaa aaaaataaaa aaaagcaatg 780 aaaaagggtt tttttaagtt gaagaccctt ttcaaatata tgttgctttg aattgtatct 840 accgtctcgt ttcttctgct ttaccgtttt tttttgcctt ctttagatat gtcttttatg 900 cttgaaaggt ccggctttaa tgcattcatc taaacgtagt attcctattt ttgaactgct 960 accaatccac catgacttta ct 982 8 905 DNA Saccharomyces cerevisiae 8 gtcaaactcc cacagccaag tccaagagta cgaaaaaaaa ctttcacaac gagttcaaaa 60 aatgtgggat gaagactccc gtttttcacc gcctagaaaa cagcgtttgc tgagaaaaaa 120 aataaatcat cgagaagaag tatgtcatca taggatgttc ccattgtaag gtgatgtgta 180 acatactcga acaaagaatg tatagagctg aatatttctc ctttaaattt caaagaaaat 240 gagaaggaaa atctcaaaca gaaacttcgt tctttttctc aagtaagcaa aagcttattg 300 agacaaagcg gaataactac gatattaata acgttgatga agctcgaaca aagttagcgt 360 cggttatgct tgcctatata aagatatatt tgccttacat tttcgttgaa cgtagaatga 420 tttttgcttt taataaattt tttgttgttc tttcagtgct tcttcaactt tgatacgaaa 480 gcaagtgcat tagtacaaca agaactggcc acaactatac tatactcatt ttttcttgcc 540 cgtgttttaa atgttttcat ccacagcatt tgatgggatg attggaagtg agacgttcga 600 gaaaatccat attttgagtc aagaattcag ataatatact gagatgatta ggtatggctg 660 ggttctacaa aaacacaaat atccggctag caatgatcac tgagcaaatt aaagcgttaa 720 ctcactcatt attgtagctt atgcgtttct cctcctctct ttttttcctc gaaccggagt 780 ggaagatcca ataacgtaat attactgatg ttgttattaa agctggcaaa aataacatga 840 ggcgtaaaac cgcactgcgg taagatgagg gtataaggtg gagatcaggc gaacaagctg 900 ttcta 905 9 908 DNA Saccharomyces cerevisiae 9 aacgatccaa tgagcgtttc atgatgccat tgtttaatca gagtgatgaa aaagaaatat 60 ttgcgacctt ttttcgttac attgatcgtg aaattttaat caaagataat ataaggacgt 120 gagatattta tctttttact tgaaattaac aatagaattg cgctaagcgg aataagagct 180 ttcgtaaacc tttctatttg caccattgcg tcaacgtata aaatggtatg acctttacac 240 aaacgcatgc ttataatctt atgtttttca tagggtgtaa tttggttgat gacgtagtct 300 aaatttgatg ctatctgcaa ttgaggtaca tataagaggt caatttcggg accaaccctt 360 ttaatcgaaa aaaacgtaat tcactagggc aagggagaac ttagcagcta atatcgtaaa 420 cctttcatac taaaaaaatg cacttaccat caacaaaaaa ctcaggacca atttccaagc 480 ttttctaggt gattgcctat aacacaaaaa gattcgctca tacatgagat ttttacatgt 540 aatagcaatt tgttccgatc agttgaaggt catcaacgca cggcaggtac atccacacct 600 atcacaaagc ccttcaataa ttcacctacg taaagttata ccgaaacatg caaaatccat 660 gaaaaattct gtatgataac gatcatatcc ttttgtattg gtggtacgat gctcaaagat 720 agttattgtt gcacctgagg caaaagcgga aatgaaaaat ccagatgggg ccaaaagcag 780 aagtattgtg tacaacaatt gcttcagcag tttaccaaac cgtttcccag caatcatcaa 840 aagttgcttt agccacattt ccgcaagata tctttgtggc tcaacgaaga gggctattcc 900 aaatgcaa 908 10 909 DNA Saccharomyces cerevisiae 10 aactttcccc cgtttaccac attgaagctg ggtgtggaag atttatttga agaaactaaa 60 acgtaccctg tcatttcctg agtccccttt caacttagtg tgaaagccga acaattataa 120 tcctcggtag acaacagatt tattgtacta aagttactct tcctgttatc ttccttgatt 180 ttactgttat agcaatgacc caccgcaatc aggagagccg ccgtatggaa tagcatacca 240 agtcataaaa tcgtcaacct attaacgggg ttcaggttct ttttcagcgt agtagccctt 300 taacaagcgc tgacaaagtt gacactcaga gaaaattcag gatttattgt aatccagcta 360 ctcatcctta gatccgcttg caggcatggt ttttttcacc ttgagaggct attttgggta 420 agccaggaag gctgaaaaat cccaaaagga cacagtaata agaaattgtt gttgttgtat 480 gatgcattta gaactcaaaa gacgagtttc tgaaaatgct tacaatactc cataggtaac 540 atgatttttt tattaaaaaa gtatactgtt cctttgggta aaaattatgc aacccttgag 600 tgtccgatga agataagact acgaaacaat ttgcggtaaa ttttttctgc tattgacatt 660 tacacatgct ccaatccatt accctttcca ttctcgtaat aaaacctcga actgttattt 720 catatttaca tctagacggg tatcggcctc aacaactcca aacaaaagta aatagaaaag 780 agccagacct atcgcaccgg gtagagccag aaaatatttt aaactatagt tgacgtattc 840 tacggctgtt gtttaggaca atactttttc cttcacaggc ttcgaattac gcacatgcag 900 aactcctgt 909 11 929 DNA Saccharomyces cerevisiae 11 gtgagacctc cggaattttg acgctgcaag tcaatctacg ggaaagaaga aattttttaa 60 acctaatgca aaataagctt ttcttggaaa ataagatttt cggcaataaa aggtaaatgc 120 agccaaaaat caaaatactt cagaagaagt cgtagcgagg actgctaagg ggaagcggat 180 ttgaagatcc tttccagaac aagaaggagc cgaaagctgt caggaactgt tcctgatttt 240 ttaggaaaac aattaatagg tatctcgtct agagtagtat ctcgagcttc cagaagttgc 300 agataatcaa aatcattgtt ttatcccttt ttttagatta cagcttagaa gagtagagag 360 caagtttact gaaacggttc cttgtttaca ataatattcc taacaaactt tacgaattag 420 gatgcagcat gattttttat attgcttcac ttcctaaagt atgaattttt atccgtagtc 480 gcaaacaaaa cagctactgg aaatctgcag cttgttaaaa accggtagtt tccgaatact 540 cctcgtcctt gagttgtata ccgttaaact tcctagggtg tcatgtgtct ggcccaattg 600 gcccacaaaa tctggtccta ttgacggttt tcttttgatt ttcagcatct tcctctaaga 660 aggacagaaa attatgtaat atatgggaga aacggcctcc caactgctaa gtgtccccgg 720 cagcacgagt aagcaaaatt caggcaaact attgcattaa gaagccgtac ataattcagc 780 gtgatatgat gaaattttgt taattgcaaa ttttagtacg atttggttgt tagtgtgtgt 840 ttatgcaagt aattattgaa ccctaagtag ttactgtctt cttttgctgt aattcgtgga 900 ttcacggccc tccagcaaca tggattgaa 929 12 900 DNA Saccharomyces cerevisiae 12 agtagcattt atgaccaaaa gcgtacttaa attagcagca aaaaaatttt taataacgaa 60 actataagga aaatacgagg tactgattat gagagtcccc gtttctcatt tttgagacat 120 gatctgaaca aggctgaaaa cagcaatctt tttcgataac ttttgcaaaa atttcaaaca 180 ttgttgtttg aatgcagcca atttttatag ggtacagagc ttaatgcttt acatgtgctt 240 tattttcggt actttcctta aagtgtctac attatctctc aggacttgaa tgtcttcggc 300 tgaattacta taaaatcttg agttttctct gaagtttaat cctaagacaa tagtggtgag 360 tgatgtagtt cacgtgtgtg ccactggtaa taatagagat aactatctca gttaagtttg 420 aaaaggtaaa aaatagttta agtagtcatt ttttgcgacg gtcattcttc tctgatgcac 480 gttctttaga ctacctataa acaccattct tacggaatta taatggaaat aaaacatcag 540 tacgtgttgc tgtcggtgat agaggggtaa cagaacctta attgaaaaat tagcacagtg 600 cataatttat taacatgatt gttttctgtg ggaaataaga aatttcagca ccagtaaaag 660 acgagaaata tagggcacat aaatgcgctc ttactcgtat gttccaggat gaaaatgttt 720 agggcatcaa gtattgccga aagggcaata tgctttaaca ccagaaaatc cactgtatac 780 tcgttacggg taaacaaagc aaaacgcagt gcgtgataat gtttctaaaa tctctgcaca 840 ctgttgaaat gcggctctga tactttagcc cttagtacct gacggtgcct aaaatgagga 900 13 951 DNA Saccharomyces cerevisiae 13 tagttggagg ttggtgagta ccagattgct tacaaaagaa tagcgagcca acatttgctc 60 tgcctcaggc ctcttggtgc tgcttgaaga ctcatcttat atggcttttg tatgtcatga 120 tttgttcttg tacattatgt gttgatatta aacaaattga tttttttttt tttgcgatag 180 caagcagata atgaaagaga caaggacttg gaacatccga taagactgcg ccgatatcga 240 tcttacagtc cttcccttgt gtcatgactt tcggaaaagc atcctcgtcg actggtagtt 300 tgctgtctgt cacgtgctga agggtctgat acattttttt aaagataaga gacggggttt 360 acccttcgga ggactaagcg agatctccaa gtaaagatct cgcttatcaa gaaagcagcc 420 aagtgtggaa cgtccttttt tttggtttca aaaagatatt caacagttta cactgcagct 480 ttaattgcct caaaaggata tcatgaggtg atctagggtc agaagggaaa gattacagca 540 tcttgagttg aatcacatct gcaaaaggtg gtattattga cgttgctctt ccttaatgga 600 aactcatggg gtttggaaag gaggtgcggt aatctatttt tttcgaacac aaaacctaac 660 cttgaaaaga aactgtccaa tttcattgaa cttacctcag aacgggccgg agtctttgct 720 ttcagtctaa catggtctaa tttcttcgaa aagcttcatt taattgttag actgtggttt 780 tacaaggaaa aaaccagtgc tatactgaag cgatacccag aactaattac cttgtgtgac 840 gattcggctc agcgaaacgg acatggtaaa attgggaatt tgaaagcagg cagcagcctt 900 gtacagcgac atgacgatag gtttagaatc cccatcacgt acgagttgaa g 951 14 1010 DNA Saccharomyces cerevisiae 14 tcctagagta gcgattcccc ttcgcgtatt cttacatctt cgaagagaac ttctggtgta 60 agtataataa atattatagc tctatcgaat ggtgcaatta tttaccaaat tctcaatagg 120 aatccataat actacatacg atactaatat tctagtattt ttatacttat tatttctttt 180 ttattacacc agcaatcgtt gcaaattatc ttctgataga atttctgagg gtatcctaaa 240 cttatgccat tttcttggac tgtaaatcat acttggatgt tgtgcattag tcaataatcg 300 gttcttgttc caacgattac atgtaaatga agggagaaat aattatggta aatcatgcgg 360 cggtcctttt ggtgatgcag tatccatagt cactacataa caatcttagt caccttgtat 420 tgattcacca cataatcctg cagagcccgc tatgtcctta atctgcgcga taactctcct 480 acccctgaat tttgagagcg ccatagcaaa ccgataaagc tggcacaatt aaaggtatcg 540 gtgttgtcag aattaggtgc ctcctgcttt tttttttttc ctgctcttat atccgttata 600 tccgaatgat ttttatcgct tgtttaaaaa atactttccc gatatatata tatagtctcc 660 ctttaaattt gtttccggta agtttttaac accaataaat gaaaagaaat gactacggtg 720 atgaatatga gccgcgcatt gaatcaggtt atgtaagtat cagaacccct aattatgatg 780 tcactcttac ccttcgatgg ctaagcggcg actgggatgc cgggaaaagc tctacaaatc 840 tactaaaaaa gtcaaatata cagctgtaaa cttctttcct cgtctacatc atggtaacga 900 ttgttcaatc tttacttcgt gtcttttttt ttttctatgt actttctatt ccaacctatg 960 tgaagactaa aattcacctt agtaaacgta aagacaatga cgataggtgc 1010 15 945 DNA Saccharomyces cerevisiae 15 acaattgcat ctgtgcttgg ctctcaagaa cggtgtttgg tgcatcaaaa gttttcgact 60 gcttatttgg tcggaaatat aaaaactcga tcctcttatc taagcagtat acattcttct 120 ttttgaaatg aatgtactcc gtaatatctt cttatttggc attttcatcc ttaacttttg 180 catggctctg aactagtcag atagttgccc ttttcagcaa acctcttatt attgaaagca 240 tggtgtacat ccgttatact attatattat aagaaattgg gatgccaatt tttttgcttt 300 tgttttgcct gttttccttc ttttcgcaaa agtaattgca gatttaatag caggatatta 360 taccgttggt aaaacttaag gattttatga acaatagctt caagtacagc attcatagaa 420 ccaactacta aggatgaaac tagtatgttt ttgtcaaaat attttcttga ccttgctgta 480 acatcaagat ctgtttctct aagatattaa agttgagtaa aaacaaagct gatatgagaa 540 aaatacgtaa ttgctccaca taatacgtgg gtcagacata aaggtagaat acttgataca 600 gaagagatta ttcggtactc ttgatggcgt gcttgaactg gtgcctctta acaaccggta 660 atatagtcag atgagtcact acgagtgtgt gtagtagcaa gtgttttacc tacgtggcag 720 taagagtagc tctatggttg tgtaatagtg gtgcttattc ctaatgctct gaagtctgaa 780 gcggtacagt tggtctggtc tatatcatgg tcaaaggagc aaacatatct tctgaagtga 840 ccgcaaatag tactatgatg tggttggcaa tataacttaa aaggaaataa ccacaaggaa 900 ttgcacccat gtacacagtt tttcccggaa attgggaaac cagta 945 16 909 DNA Saccharomyces cerevisiae 16 gcataatgcc cgctataaac cttatttttt atatgggggt ctggcgcttc gggaaaagag 60 aggaaaactt gtaactcaat atatctcgat acaacattac gttttgtaaa tttatcacaa 120 aagccaaatg atgatatctc tcttgcaagt tatcgaacat tgattggtaa tttgtttgaa 180 aattgttaat ttattgaata tttcttttgc aaaagaaata gtctcagcga aagctggtta 240 caaaatttac atcatgagtt tacgggattt gtaaatacgc tttttgcata aaaatacttt 300 gccgtttccc acccttgcat attcacttac tccccccttc atatactcta tgtaatgatg 360 attaagcttt ggccgctaag tctctcaatt agtgttgatt ttggttttat tcatatgatt 420 cttctttagt gaagtattga tcaattacgt gagtcagctt tttgaaaacc ccatttggaa 480 ggaattagga aattattttg cttactacga ccactaattt accgccattt ctgggccttt 540 ttattgacta ttttgaccat gtgctcgact agaagaacgg catcataatc tgctggtaga 600 gttagtctat aatgattgtt gaaaataaag gcataagaga tattccacct aaaattcaag 660 ttattgactt tattatcagg atcttagtat ccttttttgg taagtcatat tcaatgaact 720 aggtctcgca aactttttgt tcgaaaagcg gtagtgcata gttatgctaa ctctggatat 780 atggcataaa ccgtacaaca ctagcccatt tttttggaag tagtgagggc agctagactg 840 tatgatgaat attcgcctgc atactgagtt ttttggtcct tttttttatg tggctggcct 900 tacgatatg 909 17 944 DNA Saccharomyces cerevisiae 17 gtttagtgca cccagactcg aatcttaaat accactttac acacctacta aattttgtcc 60 tcacaaaatg aagacaggat tcaaaaccga ttaatagtag cagaaactaa aaaagtacga 120 atattagtaa aattcatgtt cttgaatcga gctactatct ttgtcgggag ggtaaacgat 180 tataactcaa aatgactgga actggtgatt attaattttt acgtttcctg tgccaataag 240 cggaagataa gaggatagaa gaaaagaaag gcggcacttg gcgaactaca atggcgatta 300 tattcatggc gattatattc atacaaaggt aatggaggcc tcggataatg gacaatattg 360 agaaaatcct tatgcttact tctcttaata aaaaatagac acagccattt attatgcgta 420 aaaaagatta cccacttgtc ttcgatgcgt gctgctgcca atcaaccttt tgagcggaac 480 ttcgagctcg caatgcgtct ggaatgttgc tagagacagt cttggttatc tgtgacatgt 540 gtttcgttca ggcgtgtgag catcttcttg ttcgatttca aaattaccgc cttgactcgt 600 gaaactggat aattcgttgg cgttttcata taagtcgtct gatggcgaaa acttttcctt 660 tacttagcat acagcaaata tccccatttg acggattttt gaaaaatgag cccgctaacc 720 cagaatgaac tgcattacca agcatttatg taaacgttcc gccaccatct ttggtaaggt 780 atactattat gttctggatt taaggttgat tcacaatttt tcatcaccaa aatctggtgg 840 catgcctagt tgtctggttt caggcaattt agccatcata gaaaagcatc ctctgtcttg 900 agttgagaaa atgttactca tagagccaaa caaataaacc ctgg 944 18 903 DNA Saccharomyces cerevisiae 18 ttcttgccgt tctcatttcc tgcacagttt ctttgattat gtttgcagaa gaatttcttt 60 atcgtttagt ctaaacaaag aattcgttgt aaagaatttg agagcggatc ttgcattttt 120 tatttatcat gcttatgttt tttctttgat gtaagaagaa gcaagtaaga tatgtgaata 180 tcttatcact aattcaaata actaagagag ctcacaacga caatttgtga cagcatgcga 240 agcaaagagc agtgatacca gtatctttca tccagtaata acatacgact gatgttatag 300 ttaaatgtta cattttgaga gacttcaacc tctcgaaacc aagaggttgg ttttaactct 360 ggtgacttca agaagggtgg gtacctttta caaagcttga gacgaagcaa tagtcagtct 420 ctgtataaca aggagaccac ctcattttcc agtaactctt gaggcatgtc ggatggtttg 480 ccttgaataa accgcagtca ttataatgaa tggcctgtac tttcaaaaca gtctggaaac 540 agaaatccat tgctgaggta ccttttagta gcactttcgt tagtgaaggt ttaaggttag 600 ttcttattta ctgcacaaga gtttacattt aaccactcta atagtaactg ttagagtggt 660 ttaactgtta ggtgatctgt tcattccatt tttcgtgttg tatctcaaga tgagatagct 720 tagcgttgct acatacataa atctaaacat ataaacacct gtgtaactcg ttaacgtctg 780 ggcttccatg cttctaccat ttagaatgat gtagaccatt tattccaaga ggataagcac 840 cctctgtgat tcaaaatgat aataagtgtt gacgacaagt tactctcgca gaattgttgt 900 caa 903 19 973 DNA Saccharomyces cerevisiae 19 tgcaatttaa agagcgtacc tgtaaataag aaggaagaac gttatgttat taatggactt 60 tttagtgtca tcgaatttta tgtaatatat aagaaggtag aataatttgg caggataatg 120 tgttagcaaa ggaggaaatc gaataccttt aaaagagaaa aaatttttta gctgcttaaa 180 tttctgtgtt ataccacccg atagattttg agttatgctt tctaattgat ctgactgcga 240 acgttttctt tatgccatct gaattgtcag gaacaaagaa gaaaaagaaa agtttttaaa 300 aaatctgtgg tcgtgtgtga tgtacctttc ctttacatgc attaatgcgc tctgaaatgt 360 ggtacgatat ccttacagag aatatatttt ctgtatatcg tgcaatgttg aataacctat 420 gaaggaaagt acccatcgct caaggtaagc attccaggag ggtcgccaga aacttaaact 480 agttttagcg acagatccga aaattgatag agacattgaa aaaatcacta ctccgtcctt 540 tttagtgctt tctcaatgca taattttggt gcacgactaa aaaattctag aacactatag 600 ttgcattttt tgggccggaa gaagaaaaac gcatgtaact ttaatgtcaa ataaagtttt 660 cacctagtaa gcgcgataca aaaaaaacac agaaatagcc ataggaaagt gaattttgtc 720 agccgactaa aattaaggtt agcttacaaa gcagcaaaaa atttgacatc gcacggtatt 780 ccctgaaaaa ggagcaggca ggtgctgtat atttttttcg gttcctgcct cttacatggc 840 gtcggtgtat cttaaatact aaagtgagct gactaccctt ttgagtgccc tatgtgacct 900 ctgatctcga aagtaaacaa gagataccta atttcacagc cacttttgtt gcggacactg 960 acgggatgtg ttg 973 20 900 DNA Saccharomyces cerevisiae 20 caaaaataac agcaagaaaa gcggaaagac catcgcaagg tggaaaggat tataatggca 60 cagcaaagtc gcacagagca ctacagtata gcatagagtg ctaatgagtt gataggccca 120 attttgatta tgccttcttt ttcatacacg acgccagagg acattattac attacagtag 180 ttcgccgcta gatgacaaac gacatcctta ccgatatgag atgtgcaaag ctacataatg 240 gcaacaagcg ttatgaacag ccttgtcttt acgaccacag aaaagccgta ttagagctct 300 tcagctgcaa aattttcttc taatatgatg caaagccatc aaaaatcatg catagttatg 360 aaatacctga tgaaacgctt cgagttcgtg ctcaagaaat tactgaaagg ttaccgagaa 420 gaaaaatatc tatgagacac gataaggccc cttctgaatc cattgtcctg ggcttgttca 480 ttctatttac cacttaaaat tgatcctttc aaaggaattt ttttctattt ccaatagtat 540 atttgtacaa aaactacaaa aatggataaa aaataacagt aatttgtgac tactgtaaat 600 atcactgatt tggattttgt aatgagtact gctcatgccc atgccgatgc aagtggatca 660 taaattttac taaacgatat tcgataatgc gccaagcctt tataaggaac tcaaaataac 720 ccatatggac agtttcagaa ggccaaataa cgatcaagga cattcactca tgtttttcaa 780 aggcgaagag tgtaaaattt tcttctatat agttcgaata ttttatctta taaatttcag 840 tcgtcatttt ccacattcga actcaaataa tgataaagaa cgctgcagta atggcttaaa 900 21 950 DNA Saccharomyces cerevisiae 21 gcaagtatgg ggtagcaagc tgcttaaagc ttcttatcac actgtaacct ctgttaaagc 60 agctgcgttg ttcttgcact ctgcacgtaa atgacgacgg ccaatgtaaa aatagcagtc 120 actgcaggcc tcttggattg tacccgtaat tacagcaatt gccattagat ttacgaagtc 180 atttaattaa atggtttgat ttatctcctt aaatgcttcc agaaatggga ctagactttt 240 ttcactcaaa cctgttcaca attattgctc tttttcaatt ataaggtaaa caaggccatc 300 tatcagcaac acagtgctcg cattttttaa ttaaactata taaaaccaac tatttgtggt 360 tgcgacttca ctttttgttg aattactacc caatcattaa tattgaagat gtgagatcat 420 agatttattg gctttgggca tctcaaatcc caagaggtca ttttaaccaa caacatttta 480 aaaagtagat ttgtctgcct cagctatgag atgcgcatgt ccctagcatc tcatatctgg 540 ttatattatt ttttccactt ggtgaatgtt gaaaaaaaca ccactcgtcc aatttatcag 600 tttgcaggtc taatgtcctt ccctgttatt taaactgtat attgtaagca tgtcttatcg 660 aaacaactta ctcagttgtc cgaaaacaaa actgcaaatt ctgtgtgtat tcacgtacta 720 gaatcctgtc aaattggatc ttgatttaag cttttatagc aacgaacttt gcatactaag 780 ttttttttgt taaccggaac tgccaagaag cattcagtaa aatacatctt catcatttac 840 tgataatact cattcagact catatcatac tatttcgaat tcattataca tcctcaaaaa 900 ccatattctt cagttgtaat aaaagataga gcctgcattt gattcgattt 950 22 967 DNA Saccharomyces cerevisiae 22 gatttaatac agtacctttc ttcgctagga tctatatgcg aatatatcac atatgtaaat 60 tataagctca tcgcaaaacc aaaaaaaaaa aaattttcaa taatttttca ctaatcttca 120 aaaacaaatg gggtaacccg tacaagagtt attaaaaccc aaaatgacaa aatcgcgaca 180 attcaatcct acttaattag caataacata ctagcggtag agctactatc acatgttgaa 240 ccttgaatgc tcaattcatt gtactcaata ctgctatcaa aagaaaaaaa atgtattaat 300 tatattcttg tcaaaatcaa ttttacacta taagaggaaa atgttcttca gtcctagtaa 360 cattagtttt ctccctttgc tagagacttt acataatatc ctagaaggta aaattcgata 420 atacagcagt aaagtcgtat attggtagca atccttggtg acgctgactt tttttttttg 480 taattttatt gtttagttca tgataaaaaa cttcaaatca cttttaatct ggtagacaga 540 gaaaacaaat cgaaacgaaa atagagaact acgaataaaa aaatataagt ggagaagatc 600 gtcactacgc attaaacaat attgatcgct caatgccagt actgcgcgta aaagtttagt 660 aacttaacga tttaggcaca atttgagaaa aatttcgccc tgcagtaagt atgttattca 720 gtacgatata aagctgaggt tttatgctgg caacgttcag attttttagg ttatcagcaa 780 tgttaaaata ttaaatagga tacttttatt gtttgagacc accctcaatg ccagatatgt 840 taaacgcttt tttctggagt gaggtatcat agaaaaaggc tcgagtacat caagcactta 900 aaggttcaac actctactgt tacttcttta agctaagcta ttcatacata atagtccatc 960 aaagtgg 967 23 981 DNA Saccharomyces cerevisiae 23 gcaatttgca gttcaacttt tcaatgatga tttagaatga tctaacactg gaagttgaag 60 tttttcaaaa atttgctgta aaatttgacc gatttgtgag attcttcctg gctgtcagaa 120 tatggggccg tagtatattg tcagacctgt tcctttaaga ggtgatggtg ataggcgttg 180 agtatgtgta gtgtttgacc cgagggtatg gttttcacaa gtactgcgca ctgtattgtg 240 aaagcagctt cggggtgcgt gattaaaaaa tgcgaccaag aataaacagg tacatcataa 300 caagggccat ttgaattgca tttatcagga tttgtaacct tgttctaaag aggcatcgta 360 tagtttaagt tcattttcca cccaatttga tgacggtgtg gaccttaacc tattgtcttg 420 aaatttaggt tatctcttag atatcacatg tgattacccc agtgaacgcg tataagctta 480 cagaaaggaa aaccggttgg ctcagtcaaa actgttgcag atttgggctc ccctgaatat 540 ttgagacatc cctaaaatga agagatatat acagctaatt ttgaatgaaa atttaaaatt 600 cgcaatgaac agtactagag atgagctttt gaagtccttt caaattattt gttcttccag 660 ttgatatttt ttattttata taccagtacc aaatataatc ttgccataca tttacctttt 720 tgaggttgtt caacggaaat ccagtgtatt tacacattct tggaaaccca tcgcttataa 780 tacgaactaa tttatttatg aacaaaggct ttggaaaagt atccctactt tttacgacgc 840 taaatcatga tacgaaactt taggaagatt aacagtcact ccataaaatc agaaagtatt 900 cgctaatagt ggaaagaaat ggttatataa agatggaaat atcttgaaag agacagttta 960 acccgaagtt ctgtcaaagt g 981 24 807 DNA Saccharomyces cerevisiae 24 ttcagaaaag caaggaaaca gtactatcgt ttagaatgta gaatgatagg ttgcttgcta 60 attctattat ggcacgaatg atacacccat attttcaaca aaatcaatac ccactagcat 120 cattgagcca actatttgtc aatgcaacca ttaccggtac ttcatcctga tttaacgagt 180 ctactttttt atcacgtcaa aatttacttg ttttcctgta aacccgaaat aaaggcaaaa 240 aagacctggg tgcaattacg aataaatgta caataatcat cctgtttgca tagtaaactt 300 ccagttagag tcacacaacg caatgaattt tgacagtttt ctgtgcgata ttctttggta 360 aacgtaaaga acaggcaact tttggtacaa tggattctag cccatatggt tcatttctgg 420 tgcattcgca aagtcagtat ttgtctagct gtgttttctg gctgagagac attatgatgt 480 tattcattgt tatggatatc tctgtagctc atgctgctta tttctcccta aaaaagtttt 540 ttctctcgaa tacattcttg accatttcat agtgaaattc ttgtacttat ttaaaaccaa 600 aaatggaagt attcatacat ccccctatca aaaacactca ataagtttcg aattattcgt 660 tcgtctaaac agtgtccaat actcaaaggg gtattcaaga cggcacaaaa tcagcatctt 720 cccttatccg tgttccagaa ataccacgct aaggtttttc ctcctacaat ccataaaatc 780 attaaggagg cagcttgaaa aatcttg 807 25 762 DNA Saccharomyces cerevisiae 25 tttctttttc cctatttctc actggttgac agaaatcagt gtgctatcat cctaccatat 60 gcgctaaact tattgtcttt ctcctcctag agatgctgta ttccatgcat attctgaacg 120 atgggttggt gtttttatca agcaaggtta atcacatggc gtggcttgct ccacacatca 180 gtagaaaacg cataccgcag cggaatcctt aaataataag tgattttact gttcatcaac 240 tacaatcgga ctctttcaca attacccttc ttgttttcca catttactgt taaatgaagg 300 gatgtacaga aggcttagga aaacctgtgc tgaatactgg atggacactg cattcccaca 360 gtgaaacttt tatagataca ctgtcagtta ttttcgaact ttcatcaagt tgctgagttt 420 tagtatccct ttgccttagc tatatgtttg aatgagcaaa atatttgcaa tgtctctagc 480 tttcttgaaa tattggttta tattgagggc ttggtaagat ttcaaatttc actttgaaat 540 actcaggaga aaaatcatgc tcttttgata atttggtgac taaacataca taaaacagtt 600 taattttggg tggtaatggc tgtgtgacta gctatagaaa gaaaaaaatt aaaaaaaaaa 660 aaaaaaatca agtagttcct gcactgcgac gtccattata gcattatgaa ttggtccctg 720 atttacgcat gcgataaact atttttagcg cagccgcata tt 762 26 874 DNA Saccharomyces cerevisiae 26 aaaatctcaa aattcccaat attcacatag tctaaagtac cgatagcaac caacatatat 60 aaacagtagt attttacgaa gctgaattgc aagattagtg agaggagaat aaccggataa 120 tttttttgga ttacgttatt gttaaaggct ataatattag gtgaaacaga atgtcctaga 180 agtttttttc tttcatgtta aatttattga ttcttgcgct tcagctttta taaaacataa 240 gaactgtttc ttcacgttaa cttcttgtgc cacatataat gatgtactag taatatgggt 300 actatttggc agatgatatt tgatttttat tcaagacggt tactgtttct acgattgata 360 ttttcattcc tggatatcat cttgccagat cacttacaat ttaggccgcg cctgaattga 420 agagtacttc aatacgtagt gtactgtcca aactctcttc caaattttta atatttagct 480 ggggttgggt aacaagtgag caagggaaaa agtgaacatt ttaagaagaa caataaaata 540 gcaagagatg gaatggtaat gcttggctct cgagaagagt agcataaaac gagacttgtt 600 taaaacagga tatgacatac ttcaattcag ctttccctat cagccgctcg agcagttata 660 taggtgtgtt gccggagtaa tttggcggag gccaacagtg gctaggcggc aacgcctgga 720 acacgcgctt aaaagttctg gaaggttcgc gaattgagaa ctgctcaggg gcgaatacag 780 gggcggcctt ggcggcaggg gggaggcctc tgtgaagtta gttatataag acttgctgtc 840 atcgtttttt tgatcccggc aggaactatc tttt 874 27 797 DNA Saccharomyces cerevisiae 27 aatagatgaa ttatgtgccg ctggacgttt atagatagca taagcacaat gacttaaagg 60 ttataatact cattgatatc actctgatta taaaatcgta atatgcgaat aggtgaacta 120 atcggaataa cccatacgac acttcaagct tcaattctat ttcaactgta gtgcctgcta 180 gtgaagaata caaaagtagc atacgtgatg tgcaaaaaat gcgctactta tcacacaagt 240 accttgcgca agaagggtac tctaaaccgg ggccatcgca ttaccagacg gagatgtatt 300 ctttatgaag caataattgg aggtgtatca agttcgaaac tgctgatgct atggatttac 360 atctttctta tgcacaaggc ttgcttgtgt ttctgagtag ttagttttta gatttttgtc 420 aagtctgggg taagttaatt cgagcaaaat taacggcacg ttattctaat gcatatgttg 480 ttcatatatt cttttacaaa gaggtttgga atgatgtcac cgatgttaga atgttaggag 540 aatttcatgt gaattttagt ccaagtgttg aagttctctt ctgcagttag ggcacgtaca 600 tggcaacgat atcgtttttg atgtattaat cttagtaggc gttgagtttg tatgttactt 660 ttctcaggtg atgaagcgtg atgacgatga caaaaatggg ttataatagg gcgcactatc 720 atcatgcgtg attgatattt aaccaatgtc ttgagtacat caactccaga aaatgggtca 780 ttatatgcct agcatgt 797 28 808 DNA Saccharomyces cerevisiae 28 tggcaatgat actcgttatt cgtaatatca gtccgtcaag gtgctgtgat ttctctattt 60 tatattgcct attatttttt caaatgattt gagccgtttt aaattgagta tgcaatgagt 120 cttttgaatc aaccgtaagg cagttccata accactgcca cgaatacgtt tcactacctt 180 gaagaatctc taatgtaggc cgtattcttc gcacttagtt ctgacgatgt agacatctca 240 ttatataaga gcataagcgc ctgtttctag aatcatttct tcgtgaccca gctttttgag 300 ttatttcgcg gtattttgaa acatttctcg agcttgacgt gaacatcctt atatttcatg 360 acaaactcga tcattggaac atccctgcct cgattttaga gctagtatca aatttcaatc 420 tctttgtgat ggagccccgc tcctatttca aaagagaagt ttcttgtatg catatgttat 480 tgaagtctga ttatagcaag tgcaatgtcg tctcaattat tttaactatt tttagccata 540 catgttagtt atcctcaaag agagcctcca gactgggaag cagtgtttgt catttcaaat 600 aagtagattt cacagtttgt atgattttcg aagccaggat tcattgggct ttgagtaaag 660 agaagccgcg tattacgaac agcttacgat attgtaaaat attcccttat tgtggtgccc 720 caatggatac atgccagaga aatgtctgtg aaattgaaca attacaatga cgagagcaag 780 taatccggcg gccttgtctc tctttcac 808 29 709 DNA Saccharomyces cerevisiae 29 agataccgtc cttggataga gcgctggaga tagctggtct caatctggtg gagtaccatg 60 ggacaccagt gatgactcta gtgacttgat cagcgggaat accagtcaac atagtggtga 120 aatcaccgta gttgaaaaca gcttcagcaa tttcaactgg gtaagtttca gttggatgag 180 cagcttggaa catatagtat tcagccaaat gagctctgat atctgagacg tagacaccta 240 attcgaccag gttaactctt tcgtcagagg gagataaagt agtggtggct ggggcagcag 300 cgacaccagc agcaatagca gcgacaccag caacaattga agttagtttg accatttttt 360 tcgattgaac ttttgtagat ctttttagtg aagatgtgag ctcactcgaa tgtaaataac 420 aatgccaaat tgtcggaaag agttaatcaa agctgctcta tttatatgcc gttttttaat 480 aagcgacgga cgaacagata aattgttgaa tagctatttc actgctgata tttctcttac 540 ttgggctccc ctatcccata ctcttcacca ctacaaatat gcagttgccc tttcttcaac 600 aatgcttttt ttatagatct cgtatacgga tccgcgcctt tgtactacct atatcttatt 660 atgatatata caggagcaca ggaatgttcg gtacagggat gataccttt 709 30 893 DNA Saccharomyces cerevisiae 30 ttgggacggt ttttgcacta agaacagacg agtttacggt tatcctcaac aagcaagcaa 60 gtatttgcta atctagatgc cattccgaat cattactcat acgttactat tgagagatgt 120 tttacaatag atgagaagaa tacaatgtcc agagctcctg gtatgctaga gtgcatattc 180 caggtcttat tcgaatcata tcataccgtc catttcaaca atggtgaaat gtggtccaca 240 tatatcagaa atcttaacat ttagtgagga gagccagtag aaaaatgtgc gcaagcggaa 300 agaagtcatt cacagacacg tttaacaaaa caccaccaca gcagctttgt ctcttgattc 360 tgatcagttt gccatcgaag aagcaaaatt gtggtgttat ttttttcaaa caaaactttt 420 ttggcaacag cagttttctt ctggatattt gtactttatc atccaaccga tgaaagctgg 480 tttcctgtca acctacattt aaatggcccg tacttcttca aaaccgctag ataagcaaat 540 taacccaact tttgagcgtc ctaaattccc cttggctcag aagactcgtt aatatgggaa 600 gtttaagtcc taccatataa tcaaattgga agctttctgt gttcgaatgg ctattctaac 660 cgctgggcta ttaatcagag gggaagtgaa atgaccgaga cgtattatac gtcatgttga 720 catcaacaat ttaaggaaaa aaataaaaaa aagcaatgaa aaagggtttt tttaagttga 780 agaccctttt caaatatatg ttgctttgaa ttgtatctac cgtctcgttt cttctgcttt 840 accgtttttt tttgccttct ttagatatgt cttttatgct tgaaaggtcc ggc 893 31 788 DNA Saccharomyces cerevisiae 31 ttcaccgcct agaaaacagc gtttgctgag aaaaaaaata aatcatcgag aagaagtatg 60 tcatcatagg atgttcccat tgtaaggtga tgtgtaacat actcgaacaa agaatgtata 120 gagctgaata tttctccttt aaatttcaaa gaaaatgaga aggaaaatct caaacagaaa 180 cttcgttctt tttctcaagt aagcaaaagc ttattgagac aaagcggaat aactacgata 240 ttaataacgt tgatgaagct cgaacaaagt tagcgtcggt tatgcttgcc tatataaaga 300 tatatttgcc ttacattttc gttgaacgta gaatgatttt tgcttttaat aaattttttg 360 ttgttctttc agtgcttctt caactttgat acgaaagcaa gtgcattagt acaacaagaa 420 ctggccacaa ctatactata ctcatttttt cttgcccgtg ttttaaatgt tttcatccac 480 agcatttgat gggatgattg gaagtgagac gttcgagaaa atccatattt tgagtcaaga 540 attcagataa tatactgaga tgattaggta tggctgggtt ctacaaaaac acaaatatcc 600 ggctagcaat gatcactgag caaattaaag cgttaactca ctcattattg tagcttatgc 660 gtttctcctc ctctcttttt ttcctcgaac cggagtggaa gatccaataa cgtaatatta 720 ctgatgttgt tattaaagct ggcaaaaata acatgaggcg taaaaccgca ctgcggtaag 780 atgagggt 788 32 825 DNA Saccharomyces cerevisiae 32 tgaaaaagaa atatttgcga ccttttttcg ttacattgat cgtgaaattt taatcaaaga 60 taatataagg acgtgagata tttatctttt tacttgaaat taacaataga attgcgctaa 120 gcggaataag agctttcgta aacctttcta tttgcaccat tgcgtcaacg tataaaatgg 180 tatgaccttt acacaaacgc atgcttataa tcttatgttt ttcatagggt gtaatttggt 240 tgatgacgta gtctaaattt gatgctatct gcaattgagg tacatataag aggtcaattt 300 cgggaccaac ccttttaatc gaaaaaaacg taattcacta gggcaaggga gaacttagca 360 gctaatatcg taaacctttc atactaaaaa aatgcactta ccatcaacaa aaaactcagg 420 accaatttcc aagcttttct aggtgattgc ctataacaca aaaagattcg ctcatacatg 480 agatttttac atgtaatagc aatttgttcc gatcagttga aggtcatcaa cgcacggcag 540 gtacatccac acctatcaca aagcccttca ataattcacc tacgtaaagt tataccgaaa 600 catgcaaaat ccatgaaaaa ttctgtatga taacgatcat atccttttgt attggtggta 660 cgatgctcaa agatagttat tgttgcacct gaggcaaaag cggaaatgaa aaatccagat 720 ggggccaaaa gcagaagtat tgtgtacaac aattgcttca gcagtttacc aaaccgtttc 780 ccagcaatca tcaaaagttg ctttagccac atttccgcaa gatat 825 33 775 DNA Saccharomyces cerevisiae 33 attgaagctg ggtgtggaag atttatttga agaaactaaa acgtaccctg tcatttcctg 60 agtccccttt caacttagtg tgaaagccga acaattataa tcctcggtag acaacagatt 120 tattgtacta aagttactct tcctgttatc ttccttgatt ttactgttat agcaatgacc 180 caccgcaatc aggagagccg ccgtatggaa tagcatacca agtcataaaa tcgtcaacct 240 attaacgggg ttcaggttct ttttcagcgt agtagccctt taacaagcgc tgacaaagtt 300 gacactcaga gaaaattcag gatttattgt aatccagcta ctcatcctta gatccgcttg 360 caggcatggt ttttttcacc ttgagaggct attttgggta agccaggaag gctgaaaaat 420 cccaaaagga cacagtaata agaaattgtt gttgttgtat gatgcattta gaactcaaaa 480 gacgagtttc tgaaaatgct tacaatactc cataggtaac atgatttttt tattaaaaaa 540 gtatactgtt cctttgggta aaaattatgc aacccttgag tgtccgatga agataagact 600 acgaaacaat ttgcggtaaa ttttttctgc tattgacatt tacacatgct ccaatccatt 660 accctttcca ttctcgtaat aaaacctcga actgttattt catatttaca tctagacggg 720 tatcggcctc aacaactcca aacaaaagta aatagaaaag agccagacct atcgc 775 34 884 DNA Saccharomyces cerevisiae 34 gctgcaagtc aatctacggg aaagaagaaa ttttttaaac ctaatgcaaa ataagctttt 60 cttggaaaat aagattttcg gcaataaaag gtaaatgcag ccaaaaatca aaatacttca 120 gaagaagtcg tagcgaggac tgctaagggg aagcggattt gaagatcctt tccagaacaa 180 gaaggagccg aaagctgtca ggaactgttc ctgatttttt aggaaaacaa ttaataggta 240 tctcgtctag agtagtatct cgagcttcca gaagttgcag ataatcaaaa tcattgtttt 300 atcccttttt ttagattaca gcttagaaga gtagagagca agtttactga aacggttcct 360 tgtttacaat aatattccta acaaacttta cgaattagga tgcagcatga ttttttatat 420 tgcttcactt cctaaagtat gaatttttat ccgtagtcgc aaacaaaaca gctactggaa 480 atctgcagct tgttaaaaac cggtagtttc cgaatactcc tcgtccttga gttgtatacc 540 gttaaacttc ctagggtgtc atgtgtctgg cccaattggc ccacaaaatc tggtcctatt 600 gacggttttc ttttgatttt cagcatcttc ctctaagaag gacagaaaat tatgtaatat 660 atgggagaaa cggcctccca actgctaagt gtccccggca gcacgagtaa gcaaaattca 720 ggcaaactat tgcattaaga agccgtacat aattcagcgt gatatgatga aattttgtta 780 attgcaaatt ttagtacgat ttggttgtta gtgtgtgttt atgcaagtaa ttattgaacc 840 ctaagtagtt actgtcttct tttgctgtaa ttcgtggatt cacg 884 35 776 DNA Saccharomyces cerevisiae 35 gtccccgttt ctcatttttg agacatgatc tgaacaaggc tgaaaacagc aatctttttc 60 gataactttt gcaaaaattt caaacattgt tgtttgaatg cagccaattt ttatagggta 120 cagagcttaa tgctttacat gtgctttatt ttcggtactt tccttaaagt gtctacatta 180 tctctcagga cttgaatgtc ttcggctgaa ttactataaa atcttgagtt ttctctgaag 240 tttaatccta agacaatagt ggtgagtgat gtagttcacg tgtgtgccac tggtaataat 300 agagataact atctcagtta agtttgaaaa ggtaaaaaat agtttaagta gtcatttttt 360 gcgacggtca ttcttctctg atgcacgttc tttagactac ctataaacac cattcttacg 420 gaattataat ggaaataaaa catcagtacg tgttgctgtc ggtgatagag gggtaacaga 480 accttaattg aaaaattagc acagtgcata atttattaac atgattgttt tctgtgggaa 540 ataagaaatt tcagcaccag taaaagacga gaaatatagg gcacataaat gcgctcttac 600 tcgtatgttc caggatgaaa atgtttaggg catcaagtat tgccgaaagg gcaatatgct 660 ttaacaccag aaaatccact gtatactcgt tacgggtaaa caaagcaaaa cgcagtgcgt 720 gataatgttt ctaaaatctc tgcacactgt tgaaatgcgg ctctgatact ttagcc 776 36 714 DNA Saccharomyces cerevisiae 36 ccagattgct tacaaaagaa tagcgagcca acatttgctc tgcctcaggc ctcttggtgc 60 tgcttgaaga ctcatcttat atggcttttg tatgtcatga tttgttcttg tacattatgt 120 gttgatatta aacaaattga tttttttttt tttgcgatag caagcagata atgaaagaga 180 caaggacttg gaacatccga taagactgcg ccgatatcga tcttacagtc cttcccttgt 240 gtcatgactt tcggaaaagc atcctcgtcg actggtagtt tgctgtctgt cacgtgctga 300 agggtctgat acattttttt aaagataaga gacggggttt acccttcgga ggactaagcg 360 agatctccaa gtaaagatct cgcttatcaa gaaagcagcc aagtgtggaa cgtccttttt 420 tttggtttca aaaagatatt caacagttta cactgcagct ttaattgcct caaaaggata 480 tcatgaggtg atctagggtc agaagggaaa gattacagca tcttgagttg aatcacatct 540 gcaaaaggtg gtattattga cgttgctctt ccttaatgga aactcatggg gtttggaaag 600 gaggtgcggt aatctatttt tttcgaacac aaaacctaac cttgaaaaga aactgtccaa 660 tttcattgaa cttacctcag aacgggccgg agtctttgct ttcagtctaa catg 714 37 757 DNA Saccharomyces cerevisiae 37 ttcgcgtatt cttacatctt cgaagagaac ttctggtgta agtataataa atattatagc 60 tctatcgaat ggtgcaatta tttaccaaat tctcaatagg aatccataat actacatacg 120 atactaatat tctagtattt ttatacttat tatttctttt ttattacacc agcaatcgtt 180 gcaaattatc ttctgataga atttctgagg gtatcctaaa cttatgccat tttcttggac 240 tgtaaatcat acttggatgt tgtgcattag tcaataatcg gttcttgttc caacgattac 300 atgtaaatga agggagaaat aattatggta aatcatgcgg cggtcctttt ggtgatgcag 360 tatccatagt cactacataa caatcttagt caccttgtat tgattcacca cataatcctg 420 cagagcccgc tatgtcctta atctgcgcga taactctcct acccctgaat tttgagagcg 480 ccatagcaaa ccgataaagc tggcacaatt aaaggtatcg gtgttgtcag aattaggtgc 540 ctcctgcttt tttttttttc ctgctcttat atccgttata tccgaatgat ttttatcgct 600 tgtttaaaaa atactttccc gatatatata tatagtctcc ctttaaattt gtttccggta 660 agtttttaac accaataaat gaaaagaaat gactacggtg atgaatatga gccgcgcatt 720 gaatcaggtt atgtaagtat cagaacccct aattatg 757 38 891 DNA Saccharomyces cerevisiae 38 ctcaagaacg gtgtttggtg catcaaaagt tttcgactgc ttatttggtc ggaaatataa 60 aaactcgatc ctcttatcta agcagtatac attcttcttt ttgaaatgaa tgtactccgt 120 aatatcttct tatttggcat tttcatcctt aacttttgca tggctctgaa ctagtcagat 180 agttgccctt ttcagcaaac ctcttattat tgaaagcatg gtgtacatcc gttatactat 240 tatattataa gaaattggga tgccaatttt tttgcttttg ttttgcctgt tttccttctt 300 ttcgcaaaag taattgcaga tttaatagca ggatattata ccgttggtaa aacttaagga 360 ttttatgaac aatagcttca agtacagcat tcatagaacc aactactaag gatgaaacta 420 gtatgttttt gtcaaaatat tttcttgacc ttgctgtaac atcaagatct gtttctctaa 480 gatattaaag ttgagtaaaa acaaagctga tatgagaaaa atacgtaatt gctccacata 540 atacgtgggt cagacataaa ggtagaatac ttgatacaga agagattatt cggtactctt 600 gatggcgtgc ttgaactggt gcctcttaac aaccggtaat atagtcagat gagtcactac 660 gagtgtgtgt agtagcaagt gttttaccta cgtggcagta agagtagctc tatggttgtg 720 taatagtggt gcttattcct aatgctctga agtctgaagc ggtacagttg gtctggtcta 780 tatcatggtc aaaggagcaa acatatcttc tgaagtgacc gcaaatagta ctatgatgtg 840 gttggcaata taacttaaaa ggaaataacc acaaggaatt gcacccatgt a 891 39 848 DNA Saccharomyces cerevisiae 39 tttttatatg ggggtctggc gcttcgggaa aagagaggaa aacttgtaac tcaatatatc 60 tcgatacaac attacgtttt gtaaatttat cacaaaagcc aaatgatgat atctctcttg 120 caagttatcg aacattgatt ggtaatttgt ttgaaaattg ttaatttatt gaatatttct 180 tttgcaaaag aaatagtctc agcgaaagct ggttacaaaa tttacatcat gagtttacgg 240 gatttgtaaa tacgcttttt gcataaaaat actttgccgt ttcccaccct tgcatattca 300 cttactcccc ccttcatata ctctatgtaa tgatgattaa gctttggccg ctaagtctct 360 caattagtgt tgattttggt tttattcata tgattcttct ttagtgaagt attgatcaat 420 tacgtgagtc agctttttga aaaccccatt tggaaggaat taggaaatta ttttgcttac 480 tacgaccact aatttaccgc catttctggg cctttttatt gactattttg accatgtgct 540 cgactagaag aacggcatca taatctgctg gtagagttag tctataatga ttgttgaaaa 600 taaaggcata agagatattc cacctaaaat tcaagttatt gactttatta tcaggatctt 660 agtatccttt tttggtaagt catattcaat gaactaggtc tcgcaaactt tttgttcgaa 720 aagcggtagt gcatagttat gctaactctg gatatatggc ataaaccgta caacactagc 780 ccattttttt ggaagtagtg agggcagcta gactgtatga tgaatattcg cctgcatact 840 gagttttt 848 40 806 DNA Saccharomyces cerevisiae 40 tgaagacagg attcaaaacc gattaatagt agcagaaact aaaaaagtac gaatattagt 60 aaaattcatg ttcttgaatc gagctactat ctttgtcggg agggtaaacg attataactc 120 aaaatgactg gaactggtga ttattaattt ttacgtttcc tgtgccaata agcggaagat 180 aagaggatag aagaaaagaa aggcggcact tggcgaacta caatggcgat tatattcatg 240 gcgattatat tcatacaaag gtaatggagg cctcggataa tggacaatat tgagaaaatc 300 cttatgctta cttctcttaa taaaaaatag acacagccat ttattatgcg taaaaaagat 360 tacccacttg tcttcgatgc gtgctgctgc caatcaacct tttgagcgga acttcgagct 420 cgcaatgcgt ctggaatgtt gctagagaca gtcttggtta tctgtgacat gtgtttcgtt 480 caggcgtgtg agcatcttct tgttcgattt caaaattacc gccttgactc gtgaaactgg 540 ataattcgtt ggcgttttca tataagtcgt ctgatggcga aaacttttcc tttacttagc 600 atacagcaaa tatccccatt tgacggattt ttgaaaaatg agcccgctaa cccagaatga 660 actgcattac caagcattta tgtaaacgtt ccgccaccat ctttggtaag gtatactatt 720 atgttctgga tttaaggttg attcacaatt tttcatcacc aaaatctggt ggcatgccta 780 gttgtctggt ttcaggcaat ttagcc 806 41 763 DNA Saccharomyces cerevisiae 41 aatttgagag cggatcttgc attttttatt tatcatgctt atgttttttc tttgatgtaa 60 gaagaagcaa gtaagatatg tgaatatctt atcactaatt caaataacta agagagctca 120 caacgacaat ttgtgacagc atgcgaagca aagagcagtg ataccagtat ctttcatcca 180 gtaataacat acgactgatg ttatagttaa atgttacatt ttgagagact tcaacctctc 240 gaaaccaaga ggttggtttt aactctggtg acttcaagaa gggtgggtac cttttacaaa 300 gcttgagacg aagcaatagt cagtctctgt ataacaagga gaccacctca ttttccagta 360 actcttgagg catgtcggat ggtttgcctt gaataaaccg cagtcattat aatgaatggc 420 ctgtactttc aaaacagtct ggaaacagaa atccattgct gaggtacctt ttagtagcac 480 tttcgttagt gaaggtttaa ggttagttct tatttactgc acaagagttt acatttaacc 540 actctaatag taactgttag agtggtttaa ctgttaggtg atctgttcat tccatttttc 600 gtgttgtatc tcaagatgag atagcttagc gttgctacat acataaatct aaacatataa 660 acacctgtgt aactcgttaa cgtctgggct tccatgcttc taccatttag aatgatgtag 720 accatttatt ccaagaggat aagcaccctc tgtgattcaa aat 763 42 873 DNA Saccharomyces cerevisiae 42 ttaatggact ttttagtgtc atcgaatttt atgtaatata taagaaggta gaataatttg 60 gcaggataat gtgttagcaa aggaggaaat cgaatacctt taaaagagaa aaaatttttt 120 agctgcttaa atttctgtgt tataccaccc gatagatttt gagttatgct ttctaattga 180 tctgactgcg aacgttttct ttatgccatc tgaattgtca ggaacaaaga agaaaaagaa 240 aagtttttaa aaaatctgtg gtcgtgtgtg atgtaccttt cctttacatg cattaatgcg 300 ctctgaaatg tggtacgata tccttacaga gaatatattt tctgtatatc gtgcaatgtt 360 gaataaccta tgaaggaaag tacccatcgc tcaaggtaag cattccagga gggtcgccag 420 aaacttaaac tagttttagc gacagatccg aaaattgata gagacattga aaaaatcact 480 actccgtcct ttttagtgct ttctcaatgc ataattttgg tgcacgacta aaaaattcta 540 gaacactata gttgcatttt ttgggccgga agaagaaaaa cgcatgtaac tttaatgtca 600 aataaagttt tcacctagta agcgcgatac aaaaaaaaca cagaaatagc cataggaaag 660 tgaattttgt cagccgacta aaattaaggt tagcttacaa agcagcaaaa aatttgacat 720 cgcacggtat tccctgaaaa aggagcaggc aggtgctgta tatttttttc ggttcctgcc 780 tcttacatgg cgtcggtgta tcttaaatac taaagtgagc tgactaccct tttgagtgcc 840 ctatgtgacc tctgatctcg aaagtaaaca aga 873 43 800 DNA Saccharomyces cerevisiae 43 ttataatggc acagcaaagt cgcacagagc actacagtat agcatagagt gctaatgagt 60 tgataggccc aattttgatt atgccttctt tttcatacac gacgccagag gacattatta 120 cattacagta gttcgccgct agatgacaaa cgacatcctt accgatatga gatgtgcaaa 180 gctacataat ggcaacaagc gttatgaaca gccttgtctt tacgaccaca gaaaagccgt 240 attagagctc ttcagctgca aaattttctt ctaatatgat gcaaagccat caaaaatcat 300 gcatagttat gaaatacctg atgaaacgct tcgagttcgt gctcaagaaa ttactgaaag 360 gttaccgaga agaaaaatat ctatgagaca cgataaggcc ccttctgaat ccattgtcct 420 gggcttgttc attctattta ccacttaaaa ttgatccttt caaaggaatt tttttctatt 480 tccaatagta tatttgtaca aaaactacaa aaatggataa aaaataacag taatttgtga 540 ctactgtaaa tatcactgat ttggattttg taatgagtac tgctcatgcc catgccgatg 600 caagtggatc ataaatttta ctaaacgata ttcgataatg cgccaagcct ttataaggaa 660 ctcaaaataa cccatatgga cagtttcaga aggccaaata acgatcaagg acattcactc 720 atgtttttca aaggcgaaga gtgtaaaatt ttcttctata tagttcgaat attttatctt 780 ataaatttca gtcgtcattt 800 44 850 DNA Saccharomyces cerevisiae 44 tctgttaaag cagctgcgtt gttcttgcac tctgcacgta aatgacgacg gccaatgtaa 60 aaatagcagt cactgcaggc ctcttggatt gtacccgtaa ttacagcaat tgccattaga 120 tttacgaagt catttaatta aatggtttga tttatctcct taaatgcttc cagaaatggg 180 actagacttt tttcactcaa acctgttcac aattattgct ctttttcaat tataaggtaa 240 acaaggccat ctatcagcaa cacagtgctc gcatttttta attaaactat ataaaaccaa 300 ctatttgtgg ttgcgacttc actttttgtt gaattactac ccaatcatta atattgaaga 360 tgtgagatca tagatttatt ggctttgggc atctcaaatc ccaagaggtc attttaacca 420 acaacatttt aaaaagtaga tttgtctgcc tcagctatga gatgcgcatg tccctagcat 480 ctcatatctg gttatattat tttttccact tggtgaatgt tgaaaaaaac accactcgtc 540 caatttatca gtttgcaggt ctaatgtcct tccctgttat ttaaactgta tattgtaagc 600 atgtcttatc gaaacaactt actcagttgt ccgaaaacaa aactgcaaat tctgtgtgta 660 ttcacgtact agaatcctgt caaattggat cttgatttaa gcttttatag caacgaactt 720 tgcatactaa gttttttttg ttaaccggaa ctgccaagaa gcattcagta aaatacatct 780 tcatcattta ctgataatac tcattcagac tcatatcata ctatttcgaa ttcattatac 840 atcctcaaaa 850 45 725 DNA Saccharomyces cerevisiae 45 gctaggatct atatgcgaat atatcacata tgtaaattat aagctcatcg caaaaccaaa 60 aaaaaaaaaa ttttcaataa tttttcacta atcttcaaaa acaaatgggg taacccgtac 120 aagagttatt aaaacccaaa atgacaaaat cgcgacaatt caatcctact taattagcaa 180 taacatacta gcggtagagc tactatcaca tgttgaacct tgaatgctca attcattgta 240 ctcaatactg ctatcaaaag aaaaaaaatg tattaattat attcttgtca aaatcaattt 300 tacactataa gaggaaaatg ttcttcagtc ctagtaacat tagttttctc cctttgctag 360 agactttaca taatatccta gaaggtaaaa ttcgataata cagcagtaaa gtcgtatatt 420 ggtagcaatc cttggtgacg ctgacttttt ttttttgtaa ttttattgtt tagttcatga 480 taaaaaactt caaatcactt ttaatctggt agacagagaa aacaaatcga aacgaaaata 540 gagaactacg aataaaaaaa tataagtgga gaagatcgtc actacgcatt aaacaatatt 600 gatcgctcaa tgccagtact gcgcgtaaaa gtttagtaac ttaacgattt aggcacaatt 660 tgagaaaaat ttcgccctgc agtaagtatg ttattcagta cgatataaag ctgaggtttt 720 atgct 725 46 881 DNA Saccharomyces cerevisiae 46 ggaagttgaa gtttttcaaa aatttgctgt aaaatttgac cgatttgtga gattcttcct 60 ggctgtcaga atatggggcc gtagtatatt gtcagacctg ttcctttaag aggtgatggt 120 gataggcgtt gagtatgtgt agtgtttgac ccgagggtat ggttttcaca agtactgcgc 180 actgtattgt gaaagcagct tcggggtgcg tgattaaaaa atgcgaccaa gaataaacag 240 gtacatcata acaagggcca tttgaattgc atttatcagg atttgtaacc ttgttctaaa 300 gaggcatcgt atagtttaag ttcattttcc acccaatttg atgacggtgt ggaccttaac 360 ctattgtctt gaaatttagg ttatctctta gatatcacat gtgattaccc cagtgaacgc 420 gtataagctt acagaaagga aaaccggttg gctcagtcaa aactgttgca gatttgggct 480 cccctgaata tttgagacat ccctaaaatg aagagatata tacagctaat tttgaatgaa 540 aatttaaaat tcgcaatgaa cagtactaga gatgagcttt tgaagtcctt tcaaattatt 600 tgttcttcca gttgatattt tttattttat ataccagtac caaatataat cttgccatac 660 atttaccttt ttgaggttgt tcaacggaaa tccagtgtat ttacacattc ttggaaaccc 720 atcgcttata atacgaacta atttatttat gaacaaaggc tttggaaaag tatccctact 780 ttttacgacg ctaaatcatg atacgaaact ttaggaagat taacagtcac tccataaaat 840 cagaaagtat tcgctaatag tggaaagaaa tggttatata a 881 

What is claimed is:
 1. A control for use in a gene expression analysis system comprising: (a) a known amount of at least one DNA selected from the group consisting of (i) SEQ ID Nos: 1-23; (ii) a degenerate variant of the sequence set forth in (i); and (iii) a complement of the sequence set forth in (i) and (ii); or (b) a known amount of at least one mRNA transcribed from the group consisting of (i) SEQ ID NOs: 24-46; (ii) a degenerate variant of the sequence set forth in (i); and (iii) a complement of the sequence set forth in (i) and (ii).
 2. A method of using a control as a negative control in a gene expression analysis system comprising: adding a known amount of said control DNA of claim 1, to a gene expression analysis system as a control sample; subjecting the sample to hybridization conditions in the absence of complementary labeled mRNA; examining the control sample for the absence or presence of signal.
 3. A method of using controls in a gene expression analysis system comprising: adding a known amount of said control DNA of claim 1, to a gene expression analysis system as a control sample; subjecting the sample to hybridization conditions in the presence of a known amount of labeled complementary mRNA of claim 1; measuring the signal values for the labeled mRNA and determining the expression level of the DNA based on the measured signal value.
 4. A method of using controls as calibrators in a gene expression analysis system comprising: adding a known amount of a said control containing known amounts of several DNAs of claim 1, to a gene expression analysis system as control samples; subjecting the samples to hybridization conditions in the presence of a said control containing known amounts of corresponding complementary labeled mRNAs of claim 1, each mRNA being at a different concentration; measuring the signal values for the labeled mRNAs and constructing a dose-response or calibration curve based on the relationship between signal value and concentration of each mRNA.
 5. A method of using controls as calibrators for gene expression ratios in a two-color gene expression analysis system comprising: adding a known amount of at least one of said controls containing a known amount of DNA of claim 1, to a two-color gene expression analysis system as control samples; subjecting the samples to hybridization conditions in the presence of a said control containing known amounts of two differently labeled corresponding complementary labeled mRNAs of claim 1, for each DNA sample present; measuring the ratio of the signal values for the two differently labeled mRNAs and comparing the signal ratio to the ratio of concentrations of the two or more differently labeled mRNAs. 